Archives of Medical Research 46 (2015) 186e192
ORIGINAL ARTICLE
Matrix Metalloproteinase 14 Overexpression Is Correlated with the Progression and Poor Prognosis of Nasopharyngeal Carcinoma Ting-hua Yan,a,* Zhong-hao Lin,b,* Jin-hua Jiang,c,d,* Sui-wan Lu,a Hua-xing Que,a Miao-an Chen,b Xiang-sheng He,a Gan-bo Que,a Ji-nan Xiao,a and Yuan-qing Chena b
a Department of Internal Medicine and Hyperbaric Oxygen, Second Hospital of Longyan City, Longyan, Fujian, China Department of Otorhinolaryngology Head and Neck Surgery, The Affiliated Mindong Hospital of Fujian Medical University, Ningde, Fujian, China c The First Clinical Medical College, Fujian Medical University, Fuzhou, Fujian, China d Department of Oncology, Longyan Humanity Hospital, Longyan, Fujian, China
Received for publication November 9, 2014; accepted March 17, 2015 (ARCMED-D-14-00647).
Background and Aims. Matrix metalloproteinase 14 (MMP14) has been identified to play a significant role in several types of cancers, but little is known about the significance of MMP14 in nasopharyngeal carcinoma (NPC) patients. The aim of this study was to explore the association of MMP14 expression with clinicopathologic features and prognosis in NPC. Methods. MMP14 mRNA and protein expressions were examined in NPC and nasopharyngeal tissues through real-time PCR and immunohistochemistry. Meanwhile, the relationship of MMP14 expression levels with clinical features and prognosis of NPC patients was analyzed. Results. MMP14 mRNA expression was markedly higher in NPC tissues than in nasopharyngeal epithelium tissues ( p 5 0.002). Using immunohistochemistry, staining for MMP14 protein was found in the normal nasopharyngeal epithelial cells and malignant epithelial cells, but increased expression of MMP14 was observed in NPC samples compared with normal nasopharyngeal epithelium samples ( p 5 0.027). In addition, high levels of MMP14 protein were positively correlated with the status of clinical stage ( p 5 0.009), N classification ( p 5 0.006), and distant metastasis ( p 5 0.005) of NPC patients. Patients with higher MMP14 expression had a significantly shorter overall survival time than did patients with low MMP14 expression. Multivariate analysis indicated that the level of MMP14 expression was an independent prognostic indicator ( p !0.001) for the survival of patients with NPC. Conclusions. MMP14 overexpression is a potentially unfavorable prognostic factor for NPC patients. Ó 2015 IMSS. Published by Elsevier Inc. Key Words: Matrix metalloproteinase-14, Nasopharyngeal carcinoma, Immunohistochemistry, Prognosis.
Introduction Nasopharyngeal carcinoma (NPC) is a head and neck malignancy disease with a distinct racial and geographical
*
These authors contributed equally to this work. Address reprint requests to: Jin-hua Jiang, The First Clinical Medical College, Fujian Medical University, Fuzhou, 350001; Department of Oncology, Longyan Humanity Hospital, Longyan, 364000, Fujian, China; Phone/Fax: þ86 0597 2180581; E-mail: freedomlongyuan@163. com
distribution. NPC is particularly common in southern China where the annual incidence rate reaches |20e50/100,000 persons (1). Unfortunately, the majority of NPC patients tend to present a more advanced stage of disease when first diagnosed because of its vague symptoms and internal location. Although NPC patients are sensitive to radiotherapy, treatment failure remains high due to the development of local recurrence and distant metastasis (2). Therefore, it is necessary to further investigate the precise molecular changes that are responsible for the progression and metastasis of NPC.
0188-4409/$ - see front matter. Copyright Ó 2015 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2015.03.006
MMP14 in Nasopharyngeal Carcinoma
Recent studies have demonstrated that matrix metalloproteinases (MMPs) in particular are responsible for the degradation of the extracellular matrix (ECM) in tumor invasion. It has been shown that specific members of the MMP not only accelerate tumor cell invasion but also alter tumor cell behavior and promote cancer progression (3). The secreted and membrane type MMPs (MTMMPs) are major members of the human MMP family. Matrix metalloproteinase-14 (MMP14) was the first membrane type MMP discovered and hence is also referred to as membrane type 1-matrix metalloproteinase (MT1-MMP). MMP14 has been suggested to be involved in many biological processes such as proliferation, invasion, angiogenesis, and basement membrane remodeling (4). Compared with benign tumor or normal tissue, increased expression of MMP14 was observed in a variety of human cancers such as lung cancer (5,6), breast cancer (7), colon cancer (7), cervical cancer (8), prostate cancer (9), and glioblastomas (10). In addition, the correlations between MMP14 expression and clinicopathological features have been explored in a variety of human carcinomas such as breast cancer (11), lung cancer (5,6), esophageal cancer (12), gastric cancer (13), cervical cancer (8), and supraglottic cancer (14), but little is known about MMP14 in NPC patients. In order to identify the role of MMP14 in the pathogenesis of NPC, we investigated the relationship of MMP14 protein expression with clinicopathological features including patient survival. We found that mRNA and protein expression levels of MMP14 were higher in NPC tissues than in nasopharyngeal tissues. Moreover, overexpression of MMP14 was associated with NPC progression and poor prognosis. Our results demonstrated that overexpressed MMP14 is a poor prognostic factor for NPC patient survival.
Materials and Methods Sample Collection A total of 20 freshly frozen NPC samples and ten normal nasopharyngeal epithelium samples were collected from the Second Hospital of Longyan City between December 2013 and June 2014. All fresh samples were immediately preserved in liquid nitrogen. One hundred and forty eight paraffin-embedded undifferentiated NPC specimens and 32 nasopharyngeal epithelium specimens were retrieved from the Second Hospital of Longyan City and Fujian Medical University between January 2005 and December 2013. No patients had received any form of tumorspecific therapy prior to diagnosis. Before the use of these clinical samples, prior consent from patients and approval from the Institutional Ethics Committee of the Second Hospital of Longyan City and Fujian Medical University
187
were obtained. The histopathological diagnosis of all samples was diagnosed by two pathologists. Clinical staging was based on the 7th edition of the AJCC Cancer Staging Manual. In the 148 NPC cases, there were 97 males and 51 females with ages ranging from 19e72 years (median 55.98 years). The clinical follow-up time of patients ranged from 9e96 months. Overall survival (OS) was defined as the interval from the date of diagnosis to NPC-related death. Real-Time Quantitative Reverse TranscriptionPolymerase Chain Reaction (RT-qPCR) To quantitate mRNA expression, total RNA was extracted from clinical samples with RNAiso Plus (Takara, Japan). The isolated total RNA was reverse transcribed using the PrimeScript RT Master Mix (Perfect Real Time) (Takara, Japan) for MMP14 according to the manufacturer instructions. Relative expression was calculated via the comparative cycle threshold (Ct) method and was normalized to the expression of GAPDH. The sequence-specific forward and reverse primers sequences for MMP14 mRNA were 50 -GGATACCCAATGCCCATTGGCCA-30 and 50 -CCTC GGTGCACCATGTTTGGC-30 , respectively. Forward and reverse primers sequences for GAPDH mRNA were 50 GCACCGTCAAGGCTGAGAAC-30 and 50 - TGGTGA AGACGCCAGTGGA -30 , respectively. qPCR was performed using SYBR Premix Ex TaqTM II (Takara, Japan) on a LightCycler (Roche Diagnostics, USA). Relative quantification of mRNA expression was calculated using the 2-DDCt method. Raw data were presented as the relative quantity of target mRNA normalized with GAPDH and relative to a calibrator sample. All qRT-PCR reactions were performed in triplicate. Immunohistochemistry Paraffin sections from NPC and nasopharyngeal epithelium specimens were deparaffinized in xylene and rehydrated in a descending ethanol series (100, 95, 90, 80, 70% ethanol) and double-distilled water according to standard protocols. Heat-induced antigen retrieval was performed in citrate buffer and boiled for 10 min. After antigen retrieval, sections were treated with 3% hydrogen peroxide and 1% bovine serum albumin to block endogenous peroxidase activity and nonspecific binding. The sections were incubated with MMP14 antibody (Abcam, clone EP1264Y, dilution 1:150) overnight at 4 C. After phosphate-buffered saline (PBS) washing, tissue sections were incubated with the biotinylated secondary antibody and streptavidine horseradish peroxidase complex, each for 20 min at room temperature. Diaminobenzidine (DAB) was used as the chromogen, and tissue sections were counterstained with hematoxylin and then viewed under a bright-field microscope.
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(weak), 2 (medium), and 3 (strong). Extent of staining was scored as 0 (0%), 1 (1e10%), 2 (10e50%), and 3 (51e100%) according to the percentages of the positive staining areas in relation to the whole carcinoma area (15). The staining index was calculated as follows: staining index 5 staining intensity proportion of positively stained tumor cells. We detected the expression of MMP14 by staining index (scored as 0, 1, 2, 3, 4, 6, or 9). For statistical analysis, final staining scores of 0e3 and 4e9 were, respectively, considered to be low and high expression. Figure 1. MMP14 mRNA expression is increased in nasopharyngeal carcinoma tissues compared with normal nasopharyngeal epithelium tissues by real-time PCR ( p !0.001).
Evaluation of Staining The tissue sections stained immunohistochemically for MMP14 were reviewed and scored separately by two pathologists blinded to the clinical parameters. Any disagreements were arbitrated by the third pathologist. For MMP14 assessment, staining intensity was scored as 0 (negative), 1
Statistical Analysis All statistical analyses were performed using SPSS v. 17.0 and GraphPad 5.0 software. Data were presented as mean SD. Unpaired t-test was applied to test the differential mRNA expression of MMP14 in NPC tissues compared to nasopharyngeal epithelium tissues. c2 test was used to examine the differences of MMP14 protein expression between normal epithelium and cancer tissues of nasopharynx. c2 test was applied to the examination of
Figure 2. Immunohistochemical staining of MMP14 in normal nasopharyngeal epithelium tissues (AeC, original magnification 400) and nasopharyngeal carcinoma tissues (DeF, original magnification 400). MMP14 staining was mostly absent in normal nasopharyngeal epithelium tissues. (A) Negative expression of MMP14 in normal epithelium (intensity score 5 0). (B) Weak expression of MMP14 in normal epithelium (intensity score 5 1). (C) Strong expression of MMP14 in normal epithelium (intensity score 5 3). (D) Negative expression of MMP14 in NPC (intensity scores 5 0). (E) Weak expression of MMP14 in NPC (intensity score 5 1). (F) Strong expression of MMP14 in NPC (intensity score 5 3). (A color figure can be found in the online version of this article.)
MMP14 in Nasopharyngeal Carcinoma
relationship between MMP14 expression levels and clinicopathologic characteristics. Survival curves were plotted using the KaplaneMeier method and compared using the log-rank test. The significance of survival variables was analyzed using the Cox multivariate proportional hazards model; p value !0.05 was considered statistically significant.
Results MMP14 mRNA and Protein Were Overexpressed in NPC Tissue In order to assess the mRNA expression levels of MMP14 in NPC, we performed real-time PCR to measure MMP14 mRNA transcripts in 20 fresh NPC tissues and ten fresh normal nasopharyngeal epithelium tissues. Compared with normal nasopharyngeal tissues, NPC tissues showed higher expression levels of MMP14 mRNA with an average increase of 3.99-fold ( p 5 0.002, Figure 1). We measured the protein expression levels of MMP14 in 148 archived paraffin-embedded NPC samples and 32 normal nasopharyngeal epithelium samples using immunohistochemical staining (Figure 2AeF). Specific MMP14 protein staining was found in normal nasopharyngeal epithelial cells and malignant epithelial cells. Furthermore, we observed that MMP14 protein was overexpressed in 55.4% (82/148) of NPC samples. In comparison, only 35.1% of normal nasopharyngeal epithelium samples had highly expressed MMP14 protein, significantly lower than that in the NPC samples ( p 5 0.027, Table 1). Correlation Between MMP14 and Clinicopathological Features in NPC Patients We next analyzed the correlation between the expression of MMP14 protein and clinicopathological characteristics of NPC. As summarized in Table 2, there were no significant correlations between MMP14 expression and gender ( p 5 0.796), age ( p 5 0.481), smoking ( p 5 0.247), pathology classification ( p 5 1.333) or T classification ( p 5 0.080). However, MMP14 overexpression was positively associated significantly with clinical stage (IeII vs. IIIeIV; p 5 0.009), N classification (N0eN1 vs. N2eN3; p 5 0.006), distant metastasis (No vs. Yes; p 5 0.005). Table 1. Protein expression of MMP14 is increased in NPC samples compared with normal nasopharyngeal samples MMP14 expression Group
Cases
High (%)
Low (%)
p
Cancer Normal
148 37
82 (55.4) 13 (35.1)
66 (44.6) 24 (64.9)
0.027
189
Survival Analysis To explore the prognostic value of MMP14 expression in NPC, we measured the association between the levels of MMP14 expression and patient survival using KaplanMeier analysis with the log-rank test. In 148 NPC patients with prognosis information, we found that MMP14 protein expression was significantly associated with the overall survival of NPC patients. Patients with lower levels of MMP14 expression had better survival than those with higher levels of MMP14 expression ( p !0.001, Figure 3). Furthermore, we also found that overexpression of MMP14 showed poor prognosis in NPC patients regardless of clinical stage, T classification, N classification and distant metastasis. Multivariate analysis showed that MMP14 overexpression was a poor independent prognostic factor for NPC patients (Table 3). Discussion MMPs are a family of at least 24 zinc-dependent endopeptidases with the collective ability to degrade the protein components of the extracellular matrix and basement membrane. MMP activity is not restricted to extracellular matrix degradation and is necessary for several processes involved in tumorigenesis, including angiogenesis, regulation of Table 2. Correlations between MMP14 protein expression and clinicopathological features in NPC MMP14 (%) Characteristics
n
Gender Female 51 Male 97 Age (years) $50 81 !50 67 Smoking No 89 Yes 59 Clinical stage IeII 59 IIIeIV 89 T classification T1eT2 87 T3eT4 61 N classification N0eN1 69 N2eN3 79 Distant metastasis No 137 Yes 11 Pathology classification DNKC 18 UDC 130
High expression
Low expression
p
29 (56.9) 53 (54.6)
22 (43.1) 44 (45.4)
0.796
47 (58.0) 35 (52.2)
34 (42.0) 32 (47.8)
0.481
54 (57.3) 28 (47.5)
35 (42.7) 31 (52.5)
0.247
25 (42.4) 57 (64.0)
34 (57.6) 32 (36.0)
0.009
43 (49.4) 39 (63.9)
44 (50.6) 22 (36.1)
0.080
30 (43.5) 52 (65.8)
39 (56.5) 27 (34.2)
0.006
71 (51.8) 11 (100)
66 (48.2) 0 (0)
0.005
7 (38.9) 75 (57.7)
11 (61.1) 55 (42.3)
0.133
DNKC, differentiated non-keratinizing carcinoma; UDC, undifferentiated carcinoma.
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Figure 3. High MMP14 protein expression predicts a poor prognosis. Patients with tumor showing a high level of MMP14 expression had a shorter survival than those with a low level of tumor MMP14 expression. The association between patient survival and MMP14 expression was estimated using the Kaplan-Meier method and the log-rank test ( p !0.001). (A color figure can be found in the online version of this article.)
growth factor and induction of the epithelialemesenchymal transition (16). The human MMP family is divided into secreted and membrane type MMPs (MT-MMPs). The type I transmembrane structural class of MT-MMPs contains MT1-MMP (MMP14), MT2-MMP (MMP15), MT3-MMP (MMP16) and MT5-MMP (MMP24). MMP14, the first MT-MMP identified, has been implicated to play an important role in tumor progression (17,18). Several members of the MMP family have been discovered they were overexpressed in NPC compared with
normal nasopharyngeal epithelium such as MMP2 (19), MMP9 (20), and MMP12 (21), but little is known about the clinical significance of MMP-14 in NPC. In our study, we first found mRNA and protein expression levels of MMP14 were higher in NPC tissues than in normal nasopharyngeal epithelial tissues. Similarly, high expression of MMP14 was observed in human lung cancer (5,6), breast cancer (7), colon cancer (7), cervical cancer (8), prostate cancer (9), and glioblastomas (10) compared with benign tumor or normal tissue. Moreover, we further found that MMP14 overexpression was significantly positively associated with clinical stages, N classification, and distant metastasis and in NPC patients. Therefore, overexpressed MMP14 in NPC may promote local cell invasion and metastasis by degrading the extracellular matrix. In vitro experiments indicated that MMP14 played a significant role in tumor progression. MMP14 was originally known as a tumor-specific activator of MMP2 (22) and is now identified to activate MMP13 and degrade a variety of ECM components including fibronectin, collagens, and laminins (23). MMP14 also processes and interacts with membrane proteins such as integrins (24), CD44 (25), syndecan-1 (26), and BRCA2 (27). It has been demonstrated that MMP14 accelerates cell migration and invasion (18,28), and serves as a collagenolytic growth factor that provides tumor cells with the space to grow but not the ability to promote proliferative responses by overturning the growth suppressive signals from 3D collagen matrix (29e31). In the past few years, overexpression of MMP14 in tumor cells has been shown to be an independent unfavorable prognostic factor in several types of human cancers. In 140 esophageal squamous cell carcinoma samples,
Table 3. Univariate and multivariate Cox regression analyses of overall survival in 148 patients with NPC Univariate analysis Parameter Gender (female vs. male) Age ($50 vs. !50 years) Smoking (no vs. yes) Clinical stage (IeII vs. IIIeIV) T classification (T1eT2 vs. T3eT4) N classification (N0eN1 vs. N2eN3) Distant metastasis (No vs. Yes) Pathology classification (DNKC vs. UDC) MMP14 (low vs. high)
p
HR
Multivariate analysis 95% CI
p
HR
95% CI
0.450
1.182
0.766e1.824
0.543
1.138
0.750e1.728
0.052
0.647
0.417e1.004
!0.001
4.431
2.727e7.201
0.888
0.934
0.362e2.412
!0.001
2.354
1.553e3.567
0.007
1.877
1.187e2.970
!0.001
7.145
4.122e12.383
!0.001
6.717
2.535e17.79
!0.001
28.786
11.607e71.388
!0.001
8.605
3.389e21.849
0.702
0.888
0.483e1.632
!0.001
3.213
2.040e5.061
!0.001
2.661
1.654e4.282
DNKC, differentiated non-keratinizing carcinoma; UDC, undifferentiatied carcinoma; HR, hazard ratio; 95% CI, 95% confidence interval.
MMP14 in Nasopharyngeal Carcinoma
advanced stage patients mostly exhibited strong or moderate immunostaining for MMP14 protein, whereas proportion of the samples with weak or no expression of MMP14w increased in early-stage patients. Furthermore, patients with high levels of MMP14 had significantly shorter overall survival time than those with low levels of MMP14 (12). Interestingly, similar results were reported in ovarian cancer. Trudel et al. (32) observed that high levels of MMP14 protein expression correlated with unfavorable prognosis, which was consistent with reports of Wang et al. (5) and Zhou et al. (6) in non-small cell lung cancer. In this study we presented the evidence that MMP14 protein expression in NPC was inversely correlated with overall patient survival. Patients with a higher expression of MMP14 protein had shorter survival time. According to multivariate analysis, overexpression of MMP14 protein was a significant predictor of poor prognosis for NPC patients. In summary, our study demonstrated that the expression of MMP14 was significantly increased in NPC and correlated with the malignant status of NPC. Moreover, our results indicated that MMP14 was a significant prognostic factor for NPC. Because of the limited patient sample size in our study, further studies are needed to strengthen these findings and to evaluate the role of MMP14 as a reliable clinical predictor of outcome for NPC patients. Acknowledgments This research was supported by grants from The Foundation of Longyan Science and Technology Program (No. 2015LY, 2013LY57) and The Foundation of Ningde Science and Technology Program (No. 20130157). Conflict of interest: The authors claim no conflict of interest.
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9. Udayakumar TS, Chen ML, Bair EL, et al. Membrane type-1-matrix metalloproteinase expressed by prostate carcinoma cells cleaves human laminin-5 beta3 chain and induces cell migration. Cancer Res 2003;63:2292e2299. 10. Munaut C, Noel A, Hougrand O, et al. Vascular endothelial growth factor expression correlates with matrix metalloproteinases MT1MMP, MMP-2 and MMP-9 in human glioblastomas. Int J Cancer 2003;106:848e855. 11. Tetu B, Brisson J, Wang CS, et al. The influence of MMP-14, TIMP-2 and MMP-2 expression on breast cancer prognosis. Breast Cancer Res 2006;8:R28. 12. Akanuma N, Hoshino I, Akutsu Y, et al. MicroRNA-133a regulates the mRNAs of two invadopodia-related proteins, FSCN1 and MMP14, in esophageal cancer. Br J Cancer 2014;110:189e198. 13. Peng CW, Wang LW, Fang M, et al. Combined features based on MT1-MMP expression, CD11b þ immunocytes density and LNR predict clinical outcomes of gastric cancer. J Transl Med 2013;11:153. 14. Zhang H, Liu M, Sun Y, et al. MMP-14 can serve as a prognostic marker in patients with supraglottic cancer. Eur Arch Otorhinolaryngol 2009;266:1427e1434. 15. Li J, Guan HY, Gong LY, et al. Clinical significance of sphingosine kinase-1 expression in human astrocytomas progression and overall patient survival. Clin Cancer Res 2008;14:6996e7003. 16. Poincloux R, Lizarraga F, Chavrier P. Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia. J Cell Sci 2009;122: 3015e3024. 17. Zarrabi K, Dufour A, Li J, et al. Inhibition of matrix metalloproteinase 14 (MMP-14)-mediated cancer cell migration. J Biol Chem 2011;286: 33167e33177. 18. Haage A, Nam DH, Ge X, et al. Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion. Biochem Biophys Res Commun 2014;450:213e218. 19. Huang T, Chen MH, Wu MY, et al. Correlation between expression of extracellular matrix metalloproteinase inducer and matrix metalloproteinase-2 and cervical lymph node metastasis of nasopharyngeal carcinoma. Ann Otol Rhinol Laryngol 2013;122:210e215. 20. Liu Z, Li L, Yang Z, et al. Increased expression of MMP9 is correlated with poor prognosis of nasopharyngeal carcinoma. BMC Cancer 2010;10:270. 21. Chung IC, Chen LC, Chung AK, et al. Matrix metalloproteinase 12 is induced by heterogeneous nuclear ribonucleoprotein K and promotes migration and invasion in nasopharyngeal carcinoma. BMC Cancer 2014;14:348. 22. Sato H, Takino T, Miyamori H. Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis. Cancer Sci 2005;96:212e217. 23. Takino T, Guo L, Domoto T, et al. MT1-MMP prevents growth inhibition by three dimensional fibronectin matrix. Biochem Biophys Res Commun 2013;436:503e508. 24. Deryugina EI, Ratnikov BI, Postnova TI, et al. Processing of integrin alpha(v) subunit by membrane type 1 matrix metalloproteinase stimulates migration of breast carcinoma cells on vitronectin and enhances tyrosine phosphorylation of focal adhesion kinase. J Biol Chem 2002; 277:9749e9756. 25. Kajita M, Itoh Y, Chiba T, et al. Membrane-type 1 matrix metalloproteinase cleaves CD44 and promotes cell migration. J Cell Biol 2001;153:893e904. 26. Endo K, Takino T, Miyamori H, et al. Cleavage of syndecan-1 by membrane type matrix metalloproteinase-1 stimulates cell migration. J Biol Chem 2003;278:40764e40770. 27. Wali N, Hosokawa K, Malik S, et al. Centrosomal BRCA2 is a target protein of membrane type-1 matrix metalloproteinase (MT1-MMP). Biochem Biophys Res Commun 2014;443:1148e1154. 28. Takino T, Watanabe Y, Matsui M, et al. Membrane-type 1 matrix metalloproteinase modulates focal adhesion stability and cell migration. Exp Cell Res 2006;312:1381e1389. 29. Hotary K, Allen E, Punturieri A, et al. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by
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membrane-type matrix metalloproteinases 1, 2, and 3. J Cell Biol 2000;149:1309e1323. 30. Hotary KB, Allen ED, Brooks PC, et al. Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix. Cell 2003;114: 33e45.
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ORIGINAL ARTICLE
Matrix Metalloproteinase 14 Overexpression Is Correlated with the Progression and Poor Prognosis of Nasopharyngeal Carcinoma Ting-hua Yan,a,* Zhong-hao Lin,b,* Jin-hua Jiang,c,d,* Sui-wan Lu,a Hua-xing Que,a Miao-an Chen,b Xiang-sheng He,a Gan-bo Que,a Ji-nan Xiao,a and Yuan-qing Chena b
a Department of Internal Medicine and Hyperbaric Oxygen, Second Hospital of Longyan City, Longyan, Fujian, China Department of Otorhinolaryngology Head and Neck Surgery, The Affiliated Mindong Hospital of Fujian Medical University, Ningde, Fujian, China c The First Clinical Medical College, Fujian Medical University, Fuzhou, Fujian, China d Department of Oncology, Longyan Humanity Hospital, Longyan, Fujian, China
Received for publication November 9, 2014; accepted March 17, 2015 (ARCMED-D-14-00647).
Background and Aims. Matrix metalloproteinase 14 (MMP14) has been identified to play a significant role in several types of cancers, but little is known about the significance of MMP14 in nasopharyngeal carcinoma (NPC) patients. The aim of this study was to explore the association of MMP14 expression with clinicopathologic features and prognosis in NPC. Methods. MMP14 mRNA and protein expressions were examined in NPC and nasopharyngeal tissues through real-time PCR and immunohistochemistry. Meanwhile, the relationship of MMP14 expression levels with clinical features and prognosis of NPC patients was analyzed. Results. MMP14 mRNA expression was markedly higher in NPC tissues than in nasopharyngeal epithelium tissues ( p 5 0.002). Using immunohistochemistry, staining for MMP14 protein was found in the normal nasopharyngeal epithelial cells and malignant epithelial cells, but increased expression of MMP14 was observed in NPC samples compared with normal nasopharyngeal epithelium samples ( p 5 0.027). In addition, high levels of MMP14 protein were positively correlated with the status of clinical stage ( p 5 0.009), N classification ( p 5 0.006), and distant metastasis ( p 5 0.005) of NPC patients. Patients with higher MMP14 expression had a significantly shorter overall survival time than did patients with low MMP14 expression. Multivariate analysis indicated that the level of MMP14 expression was an independent prognostic indicator ( p !0.001) for the survival of patients with NPC. Conclusions. MMP14 overexpression is a potentially unfavorable prognostic factor for NPC patients. Ó 2015 IMSS. Published by Elsevier Inc. Key Words: Matrix metalloproteinase-14, Nasopharyngeal carcinoma, Immunohistochemistry, Prognosis.
Introduction Nasopharyngeal carcinoma (NPC) is a head and neck malignancy disease with a distinct racial and geographical
*
These authors contributed equally to this work. Address reprint requests to: Jin-hua Jiang, The First Clinical Medical College, Fujian Medical University, Fuzhou, 350001; Department of Oncology, Longyan Humanity Hospital, Longyan, 364000, Fujian, China; Phone/Fax: þ86 0597 2180581; E-mail: freedomlongyuan@163. com
distribution. NPC is particularly common in southern China where the annual incidence rate reaches |20e50/100,000 persons (1). Unfortunately, the majority of NPC patients tend to present a more advanced stage of disease when first diagnosed because of its vague symptoms and internal location. Although NPC patients are sensitive to radiotherapy, treatment failure remains high due to the development of local recurrence and distant metastasis (2). Therefore, it is necessary to further investigate the precise molecular changes that are responsible for the progression and metastasis of NPC.
0188-4409/$ - see front matter. Copyright Ó 2015 IMSS. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.arcmed.2015.03.006
MMP14 in Nasopharyngeal Carcinoma
Recent studies have demonstrated that matrix metalloproteinases (MMPs) in particular are responsible for the degradation of the extracellular matrix (ECM) in tumor invasion. It has been shown that specific members of the MMP not only accelerate tumor cell invasion but also alter tumor cell behavior and promote cancer progression (3). The secreted and membrane type MMPs (MTMMPs) are major members of the human MMP family. Matrix metalloproteinase-14 (MMP14) was the first membrane type MMP discovered and hence is also referred to as membrane type 1-matrix metalloproteinase (MT1-MMP). MMP14 has been suggested to be involved in many biological processes such as proliferation, invasion, angiogenesis, and basement membrane remodeling (4). Compared with benign tumor or normal tissue, increased expression of MMP14 was observed in a variety of human cancers such as lung cancer (5,6), breast cancer (7), colon cancer (7), cervical cancer (8), prostate cancer (9), and glioblastomas (10). In addition, the correlations between MMP14 expression and clinicopathological features have been explored in a variety of human carcinomas such as breast cancer (11), lung cancer (5,6), esophageal cancer (12), gastric cancer (13), cervical cancer (8), and supraglottic cancer (14), but little is known about MMP14 in NPC patients. In order to identify the role of MMP14 in the pathogenesis of NPC, we investigated the relationship of MMP14 protein expression with clinicopathological features including patient survival. We found that mRNA and protein expression levels of MMP14 were higher in NPC tissues than in nasopharyngeal tissues. Moreover, overexpression of MMP14 was associated with NPC progression and poor prognosis. Our results demonstrated that overexpressed MMP14 is a poor prognostic factor for NPC patient survival.
Materials and Methods Sample Collection A total of 20 freshly frozen NPC samples and ten normal nasopharyngeal epithelium samples were collected from the Second Hospital of Longyan City between December 2013 and June 2014. All fresh samples were immediately preserved in liquid nitrogen. One hundred and forty eight paraffin-embedded undifferentiated NPC specimens and 32 nasopharyngeal epithelium specimens were retrieved from the Second Hospital of Longyan City and Fujian Medical University between January 2005 and December 2013. No patients had received any form of tumorspecific therapy prior to diagnosis. Before the use of these clinical samples, prior consent from patients and approval from the Institutional Ethics Committee of the Second Hospital of Longyan City and Fujian Medical University
187
were obtained. The histopathological diagnosis of all samples was diagnosed by two pathologists. Clinical staging was based on the 7th edition of the AJCC Cancer Staging Manual. In the 148 NPC cases, there were 97 males and 51 females with ages ranging from 19e72 years (median 55.98 years). The clinical follow-up time of patients ranged from 9e96 months. Overall survival (OS) was defined as the interval from the date of diagnosis to NPC-related death. Real-Time Quantitative Reverse TranscriptionPolymerase Chain Reaction (RT-qPCR) To quantitate mRNA expression, total RNA was extracted from clinical samples with RNAiso Plus (Takara, Japan). The isolated total RNA was reverse transcribed using the PrimeScript RT Master Mix (Perfect Real Time) (Takara, Japan) for MMP14 according to the manufacturer instructions. Relative expression was calculated via the comparative cycle threshold (Ct) method and was normalized to the expression of GAPDH. The sequence-specific forward and reverse primers sequences for MMP14 mRNA were 50 -GGATACCCAATGCCCATTGGCCA-30 and 50 -CCTC GGTGCACCATGTTTGGC-30 , respectively. Forward and reverse primers sequences for GAPDH mRNA were 50 GCACCGTCAAGGCTGAGAAC-30 and 50 - TGGTGA AGACGCCAGTGGA -30 , respectively. qPCR was performed using SYBR Premix Ex TaqTM II (Takara, Japan) on a LightCycler (Roche Diagnostics, USA). Relative quantification of mRNA expression was calculated using the 2-DDCt method. Raw data were presented as the relative quantity of target mRNA normalized with GAPDH and relative to a calibrator sample. All qRT-PCR reactions were performed in triplicate. Immunohistochemistry Paraffin sections from NPC and nasopharyngeal epithelium specimens were deparaffinized in xylene and rehydrated in a descending ethanol series (100, 95, 90, 80, 70% ethanol) and double-distilled water according to standard protocols. Heat-induced antigen retrieval was performed in citrate buffer and boiled for 10 min. After antigen retrieval, sections were treated with 3% hydrogen peroxide and 1% bovine serum albumin to block endogenous peroxidase activity and nonspecific binding. The sections were incubated with MMP14 antibody (Abcam, clone EP1264Y, dilution 1:150) overnight at 4 C. After phosphate-buffered saline (PBS) washing, tissue sections were incubated with the biotinylated secondary antibody and streptavidine horseradish peroxidase complex, each for 20 min at room temperature. Diaminobenzidine (DAB) was used as the chromogen, and tissue sections were counterstained with hematoxylin and then viewed under a bright-field microscope.
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(weak), 2 (medium), and 3 (strong). Extent of staining was scored as 0 (0%), 1 (1e10%), 2 (10e50%), and 3 (51e100%) according to the percentages of the positive staining areas in relation to the whole carcinoma area (15). The staining index was calculated as follows: staining index 5 staining intensity proportion of positively stained tumor cells. We detected the expression of MMP14 by staining index (scored as 0, 1, 2, 3, 4, 6, or 9). For statistical analysis, final staining scores of 0e3 and 4e9 were, respectively, considered to be low and high expression. Figure 1. MMP14 mRNA expression is increased in nasopharyngeal carcinoma tissues compared with normal nasopharyngeal epithelium tissues by real-time PCR ( p !0.001).
Evaluation of Staining The tissue sections stained immunohistochemically for MMP14 were reviewed and scored separately by two pathologists blinded to the clinical parameters. Any disagreements were arbitrated by the third pathologist. For MMP14 assessment, staining intensity was scored as 0 (negative), 1
Statistical Analysis All statistical analyses were performed using SPSS v. 17.0 and GraphPad 5.0 software. Data were presented as mean SD. Unpaired t-test was applied to test the differential mRNA expression of MMP14 in NPC tissues compared to nasopharyngeal epithelium tissues. c2 test was used to examine the differences of MMP14 protein expression between normal epithelium and cancer tissues of nasopharynx. c2 test was applied to the examination of
Figure 2. Immunohistochemical staining of MMP14 in normal nasopharyngeal epithelium tissues (AeC, original magnification 400) and nasopharyngeal carcinoma tissues (DeF, original magnification 400). MMP14 staining was mostly absent in normal nasopharyngeal epithelium tissues. (A) Negative expression of MMP14 in normal epithelium (intensity score 5 0). (B) Weak expression of MMP14 in normal epithelium (intensity score 5 1). (C) Strong expression of MMP14 in normal epithelium (intensity score 5 3). (D) Negative expression of MMP14 in NPC (intensity scores 5 0). (E) Weak expression of MMP14 in NPC (intensity score 5 1). (F) Strong expression of MMP14 in NPC (intensity score 5 3). (A color figure can be found in the online version of this article.)
MMP14 in Nasopharyngeal Carcinoma
relationship between MMP14 expression levels and clinicopathologic characteristics. Survival curves were plotted using the KaplaneMeier method and compared using the log-rank test. The significance of survival variables was analyzed using the Cox multivariate proportional hazards model; p value !0.05 was considered statistically significant.
Results MMP14 mRNA and Protein Were Overexpressed in NPC Tissue In order to assess the mRNA expression levels of MMP14 in NPC, we performed real-time PCR to measure MMP14 mRNA transcripts in 20 fresh NPC tissues and ten fresh normal nasopharyngeal epithelium tissues. Compared with normal nasopharyngeal tissues, NPC tissues showed higher expression levels of MMP14 mRNA with an average increase of 3.99-fold ( p 5 0.002, Figure 1). We measured the protein expression levels of MMP14 in 148 archived paraffin-embedded NPC samples and 32 normal nasopharyngeal epithelium samples using immunohistochemical staining (Figure 2AeF). Specific MMP14 protein staining was found in normal nasopharyngeal epithelial cells and malignant epithelial cells. Furthermore, we observed that MMP14 protein was overexpressed in 55.4% (82/148) of NPC samples. In comparison, only 35.1% of normal nasopharyngeal epithelium samples had highly expressed MMP14 protein, significantly lower than that in the NPC samples ( p 5 0.027, Table 1). Correlation Between MMP14 and Clinicopathological Features in NPC Patients We next analyzed the correlation between the expression of MMP14 protein and clinicopathological characteristics of NPC. As summarized in Table 2, there were no significant correlations between MMP14 expression and gender ( p 5 0.796), age ( p 5 0.481), smoking ( p 5 0.247), pathology classification ( p 5 1.333) or T classification ( p 5 0.080). However, MMP14 overexpression was positively associated significantly with clinical stage (IeII vs. IIIeIV; p 5 0.009), N classification (N0eN1 vs. N2eN3; p 5 0.006), distant metastasis (No vs. Yes; p 5 0.005). Table 1. Protein expression of MMP14 is increased in NPC samples compared with normal nasopharyngeal samples MMP14 expression Group
Cases
High (%)
Low (%)
p
Cancer Normal
148 37
82 (55.4) 13 (35.1)
66 (44.6) 24 (64.9)
0.027
189
Survival Analysis To explore the prognostic value of MMP14 expression in NPC, we measured the association between the levels of MMP14 expression and patient survival using KaplanMeier analysis with the log-rank test. In 148 NPC patients with prognosis information, we found that MMP14 protein expression was significantly associated with the overall survival of NPC patients. Patients with lower levels of MMP14 expression had better survival than those with higher levels of MMP14 expression ( p !0.001, Figure 3). Furthermore, we also found that overexpression of MMP14 showed poor prognosis in NPC patients regardless of clinical stage, T classification, N classification and distant metastasis. Multivariate analysis showed that MMP14 overexpression was a poor independent prognostic factor for NPC patients (Table 3). Discussion MMPs are a family of at least 24 zinc-dependent endopeptidases with the collective ability to degrade the protein components of the extracellular matrix and basement membrane. MMP activity is not restricted to extracellular matrix degradation and is necessary for several processes involved in tumorigenesis, including angiogenesis, regulation of Table 2. Correlations between MMP14 protein expression and clinicopathological features in NPC MMP14 (%) Characteristics
n
Gender Female 51 Male 97 Age (years) $50 81 !50 67 Smoking No 89 Yes 59 Clinical stage IeII 59 IIIeIV 89 T classification T1eT2 87 T3eT4 61 N classification N0eN1 69 N2eN3 79 Distant metastasis No 137 Yes 11 Pathology classification DNKC 18 UDC 130
High expression
Low expression
p
29 (56.9) 53 (54.6)
22 (43.1) 44 (45.4)
0.796
47 (58.0) 35 (52.2)
34 (42.0) 32 (47.8)
0.481
54 (57.3) 28 (47.5)
35 (42.7) 31 (52.5)
0.247
25 (42.4) 57 (64.0)
34 (57.6) 32 (36.0)
0.009
43 (49.4) 39 (63.9)
44 (50.6) 22 (36.1)
0.080
30 (43.5) 52 (65.8)
39 (56.5) 27 (34.2)
0.006
71 (51.8) 11 (100)
66 (48.2) 0 (0)
0.005
7 (38.9) 75 (57.7)
11 (61.1) 55 (42.3)
0.133
DNKC, differentiated non-keratinizing carcinoma; UDC, undifferentiated carcinoma.
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Figure 3. High MMP14 protein expression predicts a poor prognosis. Patients with tumor showing a high level of MMP14 expression had a shorter survival than those with a low level of tumor MMP14 expression. The association between patient survival and MMP14 expression was estimated using the Kaplan-Meier method and the log-rank test ( p !0.001). (A color figure can be found in the online version of this article.)
growth factor and induction of the epithelialemesenchymal transition (16). The human MMP family is divided into secreted and membrane type MMPs (MT-MMPs). The type I transmembrane structural class of MT-MMPs contains MT1-MMP (MMP14), MT2-MMP (MMP15), MT3-MMP (MMP16) and MT5-MMP (MMP24). MMP14, the first MT-MMP identified, has been implicated to play an important role in tumor progression (17,18). Several members of the MMP family have been discovered they were overexpressed in NPC compared with
normal nasopharyngeal epithelium such as MMP2 (19), MMP9 (20), and MMP12 (21), but little is known about the clinical significance of MMP-14 in NPC. In our study, we first found mRNA and protein expression levels of MMP14 were higher in NPC tissues than in normal nasopharyngeal epithelial tissues. Similarly, high expression of MMP14 was observed in human lung cancer (5,6), breast cancer (7), colon cancer (7), cervical cancer (8), prostate cancer (9), and glioblastomas (10) compared with benign tumor or normal tissue. Moreover, we further found that MMP14 overexpression was significantly positively associated with clinical stages, N classification, and distant metastasis and in NPC patients. Therefore, overexpressed MMP14 in NPC may promote local cell invasion and metastasis by degrading the extracellular matrix. In vitro experiments indicated that MMP14 played a significant role in tumor progression. MMP14 was originally known as a tumor-specific activator of MMP2 (22) and is now identified to activate MMP13 and degrade a variety of ECM components including fibronectin, collagens, and laminins (23). MMP14 also processes and interacts with membrane proteins such as integrins (24), CD44 (25), syndecan-1 (26), and BRCA2 (27). It has been demonstrated that MMP14 accelerates cell migration and invasion (18,28), and serves as a collagenolytic growth factor that provides tumor cells with the space to grow but not the ability to promote proliferative responses by overturning the growth suppressive signals from 3D collagen matrix (29e31). In the past few years, overexpression of MMP14 in tumor cells has been shown to be an independent unfavorable prognostic factor in several types of human cancers. In 140 esophageal squamous cell carcinoma samples,
Table 3. Univariate and multivariate Cox regression analyses of overall survival in 148 patients with NPC Univariate analysis Parameter Gender (female vs. male) Age ($50 vs. !50 years) Smoking (no vs. yes) Clinical stage (IeII vs. IIIeIV) T classification (T1eT2 vs. T3eT4) N classification (N0eN1 vs. N2eN3) Distant metastasis (No vs. Yes) Pathology classification (DNKC vs. UDC) MMP14 (low vs. high)
p
HR
Multivariate analysis 95% CI
p
HR
95% CI
0.450
1.182
0.766e1.824
0.543
1.138
0.750e1.728
0.052
0.647
0.417e1.004
!0.001
4.431
2.727e7.201
0.888
0.934
0.362e2.412
!0.001
2.354
1.553e3.567
0.007
1.877
1.187e2.970
!0.001
7.145
4.122e12.383
!0.001
6.717
2.535e17.79
!0.001
28.786
11.607e71.388
!0.001
8.605
3.389e21.849
0.702
0.888
0.483e1.632
!0.001
3.213
2.040e5.061
!0.001
2.661
1.654e4.282
DNKC, differentiated non-keratinizing carcinoma; UDC, undifferentiatied carcinoma; HR, hazard ratio; 95% CI, 95% confidence interval.
MMP14 in Nasopharyngeal Carcinoma
advanced stage patients mostly exhibited strong or moderate immunostaining for MMP14 protein, whereas proportion of the samples with weak or no expression of MMP14w increased in early-stage patients. Furthermore, patients with high levels of MMP14 had significantly shorter overall survival time than those with low levels of MMP14 (12). Interestingly, similar results were reported in ovarian cancer. Trudel et al. (32) observed that high levels of MMP14 protein expression correlated with unfavorable prognosis, which was consistent with reports of Wang et al. (5) and Zhou et al. (6) in non-small cell lung cancer. In this study we presented the evidence that MMP14 protein expression in NPC was inversely correlated with overall patient survival. Patients with a higher expression of MMP14 protein had shorter survival time. According to multivariate analysis, overexpression of MMP14 protein was a significant predictor of poor prognosis for NPC patients. In summary, our study demonstrated that the expression of MMP14 was significantly increased in NPC and correlated with the malignant status of NPC. Moreover, our results indicated that MMP14 was a significant prognostic factor for NPC. Because of the limited patient sample size in our study, further studies are needed to strengthen these findings and to evaluate the role of MMP14 as a reliable clinical predictor of outcome for NPC patients. Acknowledgments This research was supported by grants from The Foundation of Longyan Science and Technology Program (No. 2015LY, 2013LY57) and The Foundation of Ningde Science and Technology Program (No. 20130157). Conflict of interest: The authors claim no conflict of interest.
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