doi: 10.1111/jop.12154
J Oral Pathol Med (2014) 43: 545–553 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Calcifying Cystic Odontogenic Tumour: immunohistochemical expression of matrix metalloproteinases, their inhibitors (TIMPs and RECK) and inducer (EMMPRIN) F abio C. Prosd ocimi1, Camila O. Rodini2, Mari C. Sogayar3, Suzana C. O. M. Sousa1, Flavia C. A. Xavier4, Kati ucia B. S. Paiva1,5 1
Department of Oral Pathology, Dental School, University of S~ ao Paulo, S~ ao Paulo, Brazil; 2Laboratory of Histology, Department of Biological Sciences, Bauru Dental School, University of S~ ao Paulo Bauru, S~ ao Paulo, Brazil; 3Cell and Molecular Therapy Center (NUCEL), Department of Biochemistry, Chemistry Institute, University of S~ ao Paulo, S~ ao Paulo, Brazil; 4Department of Propedeutics and 5 Integrated Clinic, Dental School, Federal University of Bahia, Salvador, Brazil; Extracellular Matrix Biology and Cellular Interaction Group, Laboratory of Molecular Mechanisms of Cytoprotection, Department of Biochemistry, Chemistry Institute, University of S~ ao Paulo, S~ ao Paulo, Brazil
BACKGROUND: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT. MATERIALS AND METHODS: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments. RESULTS: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003).
Correspondence: Kati ucia B. S. Paiva, Department of Oral Pathology, Dental School, University of S~ao Paulo, Avenida Lineu Prestes, 748 bloco 9 superior, sala 959/974/976, Cidade Universitaria, S~ao Paulo, SP 05508000, Brazil. Tel/Fax: +551130913820, E-mail: [email protected] Accepted for publication December 21, 2013
CONCLUSIONS: We concluded that these proteins/ enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour. J Oral Pathol Med (2014) 43: 545–553 Keywords: Calcifying Cystic Odontogenic Tumour; EMMPRIN; matrix metalloproteinases; RECK; tissue inhibitors of MMPs
Introduction After the first description of the currently named calcifying cystic odontogenic tumour (CCOT) as a benign odontogenic cyst in 1962 by Gorlin and colleagues (1), the question on the cystic or neoplastic nature of this lesion raised several mode of classifications. Then, considering the different histomorphological and clinicopathological features of this set of tumours, the World Health Organization (WHO) in 2005 renamed these lesions as tumours exhibiting three variants (2): (I) calcifying cystic odontogenic tumour (CCOT) defined as a benign cystic neoplasm characterized by an ameloblastoma-like epithelium with ghost cells that may calcify; (II) dentinogenic ghost cell tumour (DGCT) defined as a locally invasive neoplasm characterized by ameloblastoma-like islands of epithelial cells in a mature connective tissue stroma; and (III) ghost cell odontogenic carcinoma (GCOC) defined as an uncommon malignant neoplasm that exhibits prominent mitotic activity, nuclear atypia and cellular pleomorphism, groups of ghost epithelial cells, necrosis, sometimes scarce mineralized or osteodentine-like (‘dentinoid’) material and infiltrative growth
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Table 1 Primary antibodies applied for immunohistochemistry Antibody
Clonality
Specificity
Source
Cataloge
MMP-2 MMP-7 MMP-9 MMP-14 TIMP-2 TIMP-3 TIMP-4 RECK EMMPRIN
Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Rabbit monoclonal Mouse monoclonal Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Rabbit polyclonal
Pro- and active forms Pro-form Pro-form Active form C-terminal region C-terminal region C-terminal region Total protein C-terminal region
Abcam Novus Biologicals Abcam Novus Biologicals Calbiochem Abcam Abcam Cell signalling Abcam
ab37150 NB600-1153 ab76003 NB110-57216 IM56 ab39185 ab58425 3433 ab70062
pattern (3). Thus, CCOT was classified as an epithelial odontogenic tumour with a contribution from odontogenic ectomesenchyme, showing histological evidence of an inductive change (4). Matrix metalloproteinases (MMPs) are the most important enzyme family responsible collectively for cleavage and degradation of all components of the extracellular matrix (ECM) and then generating bioactive molecules. Several pericellular and extracellular proteins are degraded or processed by MMPs (5). Nowadays, the proteolytic target spectrum of MMPs was amplified, including other molecules on cell membrane and pericellular proteins non-related to ECM (other proteinases, intracellular substrates, latent growth factors, proteinase inhibitors, chemostatic molecules, growth factor binding proteins, cell membrane receptor, cell–cell and ECM–cell adhesion molecules), leading to the regulation of cell behaviour in several pathways, mainly signal transduction (6–10). Mammalian MMPs (24 members) are classified in soluble and cell membrane anchor-MMP (MT-MMP) in relation to enzyme-substrate in vitro specificity and molecular structure, as well as discovery chronology. MMPs are divided in collagenases (MMP-1, -8 and -13), gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), matrilysins (MMP-7 and -26), MT-MMPs (MMP-14, -15, -16, 1-7, -24 and -25) and heterogeneous group (MMP-12, -19, -20, -21, -23, 27 and -28). Mostly, soluble MMPs are secreted as pro-enzymes, and activation is requested into ECM (11), whereas MT-MMPs are activated in intracellular space and expressed on cell membrane as active form (12). MMPs are regulated at transcriptional and post-transcriptional levels by their inhibitors and inductors, as well as interaction with specific ECM components (13). Gene expression is controlled by several stimulant and suppressor factors that influence many signalling pathways, such as integrins, ECM proteins, hormones, growth factors, oncogenes, cytokines, epigenetic regulation, cell stress, morphological changes and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) (14, 15). Regarding enzymatic inhibition, pro-MMP and its active form can be inhibited in the ECM by their inhibitors anchored on the cell membrane as reversion-inducing cysteine-rich protein with Kazal motifs (RECK) (16) and secreted into the ECM as tissue inhibitor of MMPs (TIMPs) (17). Most MMPs play central role in physiological conditions (stem cell differentiation, proliferation, cell mobility, tissue remodelling, wound J Oral Pathol Med
Dilution 1:250 1:500 1:200 1:500 1:500 1:200 1:200 1:50 1:500
healing, angiogenesis and apoptosis), and an imbalance between MMPs, their inhibitors and inducer is implicated in different pathological processes (cancer, degenerative diseases) through several mechanisms, mainly related to tissue destruction, fibrosis and changes in the microenvironment (ECM composition). MMP overexpression has already been correlated with tumoural aggressiveness, stage and prognosis in a large range of malignant tumours (18–20); however, little is known about ECM changes and cell–ECM interactions in odontogenic tumours. Thus, our aim was to evaluate the immunoexpression of some MMPs (-2, -7, -9 and -14), TIMPs (-2, -3 and -4), RECK and EMMPRIN in epithelial and stromal compartments of CCOT.
Material and methods Patient tissue samples The research was approved by the Ethics Committee of the Dental School, University of S~ao Paulo. Eighteen cases of calcifying cystic odontogenic tumour (CCOT), intraosseous and without other associated odontogenic tumours were obtained from the files of the Pathology Laboratory, Department of Stomatology (from 2000 to 2012). Histopathological diagnosis was confirmed by an oral pathologist
Table 2 Main clinical data of Calcifying Cystic Odontogenic Tumour cases in the studied sample Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Gender Male Male Male Female Female Male Female Male Female Female Female Male Female Female Male Male Male Male
Age (years)
Site
39 40 36 21 27 28 26 19 14 20 20 26 28 21 47 39 15 25
Maxilla Maxilla Maxilla Maxilla Mandible Uninformed Anterior maxilla Anterior maxilla Right maxilla Anterior maxilla Anterior maxilla Right angle mandible Uninformed Maxilla Mandible Mandible Right maxilla Anterior maxilla
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(S.O.M.S.) through the slides review stained with haematoxylin and eosin, according to the WHO classification of head and neck tumours (3). The main clinical data as gender, age and tumour site were also collected.
3% H2O2 solution for 45 min, and the free charges were neutralized by 1% borax solution for 15 min. Heat-induced epitope retrieval (HIER) was performed with phosphatecitrate buffer – 93–96°C, pH 6.0 for 20 min (P4809; SigmaAldrich, St. Louis, MO, USA – except for MMP-2, for which no epitope retrieval was recommended by the manufacturer), and non-specific binding was blocked by Protein Block Serum-Free (X0909; DAKO, Carpinteria, CA, USA) for 10 min. The sections were incubated with primary antibodies (Table 1) in a humid chamber at 4°C
Immunohistochemistry Formalin-fixed paraffin-embedded (FFPE) CCOT tissue sections (4 lm thickness) were mounted on silane-coated slides. After deparaffinization and rehydration in ethanolgraded series, the endogenous peroxidase was blocked by A
B
C
D
E
F
G
H
547
Figure 1 Immunoexpression of matrix metalloproteinases 2, 7, 9 and 14 in calcifying cystic odontogenic tumour. Bl: basal layer, CV: capillary vessels, ECM: extracellular matrix, Ep: epithelial layer, IC: inflammatory cells, GC: ghost cells, MC: mesenchymal cells, SC: suprabasal spindle and stellate cells, St: stromal layer. Bar: 50 and 200 lm. J Oral Pathol Med
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overnight, and detection was performed using EnVisionTM + Dual Link System-HRP (K4061; DAKO) or LSAB + System-HRP (K0690; DAKO). The immunostaining was revealed by Liquid DAB+Substrate-Chromogen System (K3468; DAKO), and the sections were counterstained with Mayer′s haematoxylin. All rinses were performed using 0.1% Triton X-100/PBS. Antibody diluent with background reducing components (S3022; DAKO) was used for both primary antibody dilution and negative control (primary antibody was replaced by this solution). Human oral
squamous cell carcinoma biopsy was used as positive control for all antibodies. Interpretation of immunohistochemical staining The immunohistochemical analysis was performed by two trained examiners (F.C.A.X. and C.O.R.) at different times. Semi-quantitative analysis of immunostained cells in both neoplastic odontogenic epithelium and adjacent stroma was performed according to immunostaining intensity: no staining ( ), weak (+), moderate (++) and intense (+++). The A
B
C
D
E
F
G
H
Figure 2 Immunoexpression of tissue inhibitors of matrix metalloproteinases (TIMPs 2, 3 and 4) and RECK in calcifying cystic odontogenic tumour. Bl: basal layer, CV: capillary vessels, ECM: extracellular matrix, Ep: epithelial layer, GC: ghost cells, MC: mesenchymal cells, SC: suprabasal spindle and stellate cells, St: stromal layer. Bar: A: 100 lm; B, D, F, H: 50 lm; C, E, G: 200 lm. J Oral Pathol Med
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percentage of immunopositive cells restricted to the neoplastic odontogenic epithelium was estimated in 2009 microscopic magnification and scored as 1 (≤ 10% of stained epithelial cells), 2 (> 10 ≤ 50%), 3 (> 50 ≤ 75%) and 4 (> 75%). Immunostaining distribution was described within epithelium (basal layer, suprabasal fusiform, stellate and ghost cells) and within stroma (mesenchymal and endothelial cells and extracellular matrix) and regarding cellular compartment as cell membrane, cytoplasmic and/or nuclear for all molecules. Statistical analysis Statistical analysis was accomplished using the statistical package Statistical Package for Social Sciences (SPSS), version 20.0 (IBM Corp., Armonk, NY, USA). Comparison of absence (negative cases – no detected staining) or presence (all positive cases independently of immunostaining intensity) found for each protein assessed between tissue compartments (neoplastic odontogenic epithelium or adjacent stroma) was performed using Fisher’s exact test, while the correlation between proteins expression within tissue compartments was performed using Spearman’s rank correlation coefficient test. For both tests, P-value < 0.05 was considered to indicate statistical significance.
Results Clinical data demonstrated that the gender distribution of CCOT was almost equal between males and females (10:8), and the individuals were usually young to middle-aged (age mean 27.7, SD = 9.36). The maxilla, predominantly the anterior region, was in fact the most affected site (Table 2). Immunohistochemical evaluation showed that all enzymes/proteins were expressed in variable amounts in both neoplastic odontogenic epithelium and adjacent stroma in all cases of CCOT. Regarding epithelial immunostaining, wide positivity for MMP-2 (n = 18/18) (Fig. 1A, B), MMP7 (n = 18/18) (Fig. 1C, D), MMP-14 (n = 18/18) (Fig. 1G, H), TIMP-3 (n = 18/18) (Fig. 2C, D), TIMP-4 (n = 18/18) (Fig. 2E, F), RECK (n = 14/18) (Fig. 2G, H) and EMMPRIN (n = 14/18) (Fig. 3A, B) was found in basal, spindle suprabasal and stellate epithelial cells surrounding ghost cells as well as ghost cells themselves. MMP-9 (n = 16/18)
A
(Fig. 1C, D) and TIMP-2 (n = 15/18) (Fig. 2A, B) were only seen in ghost cells (Table 3). Cytoplasmic immunostaining alone was found for MMP-2, MMP-7 and TIMP-3, while simultaneous cytoplasmic and cell membrane immunostaining were found for MMP-14, RECK and EMMPRIN. Only TIMP-4 showed both cytoplasmic and nuclear immunostaining patterns (Table 4). Concerning the percentage of neoplastic epithelial immunopositive cells in CCOT, MMP-2, MMP-7, MMP-14 and TIMP-3 presented score 3, while TIMP-4 showed score 4. On the other hand, scarce immunostaining corresponding to score 2 was observed for MMP-9, TIMP-2, RECK and EMMPRIN in most cases (Table 4). Regarding immunostaining intensity, cells were intense or moderately stained for MMP-2 (n = 18/18), MMP-7 (n = 12/18), MMP-9 (n = 11/18), MMP-14 (n = 13/18), TIMP-3 (n = 16/18) and TIMP-4 (n = 18/18), whereas weak staining was observed for TIMP-2 (n = 13/18), RECK (n = 11/18) and EMMPRIN (n = 8/18) in most cases (Table 4). When adjacent stroma was analysed, both mesenchymal and endothelial cells, as well as the ECM, were also immunopositive for MMP-2, MMP-7, TIMP-3, TIMP-4. MMP-9, TIMP-2, while positivity for RECK was only seen within mesenchymal cells, and MMP-14 was restricted to endothelial cells (Table 5). Cytoplasmic immunostaining alone was detected for MMP-2, MMP-7, MMP-9 and TIMP-2, and cell membrane immunostaining alone was seen for MMP-14. Simultaneous cytoplasmic and nuclear immunostaining were found for TIMP-4, and both cytoplasmic and cell membrane immunostaining were found for RECK and EMMPRIN. Regarding immunostaining intensity, intense or moderate staining was observed for MMP-2, MMP-7, MMP-14, TIMP-2, TIMP-3 and TIMP-4, whereas weak staining for MMP-9 was observed in most cases. For RECK and EMMPRIN, only a few cases presented weak positivity (n = 3 and n = 2, respectively) (Tables 3 and 5). Comparing the immunostaining positivity for each protein between tissue compartments (neoplastic odontogenic epithelium and adjacent stroma), a statistically significant positivity for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001) was found within the neoplastic odontogenic epithelium (Table 4). Furthermore, strong positive correlation between stromal EMMPRIN and stromal RECK expression was found (P = 0.003 and
549
B
Figure 3 Immunoexpression of inducer of matrix metalloproteinases (EMMPRIN) in calcifying cystic odontogenic tumour. Bl: basal layer, CV: capillary vessels, ECM: extracellular matrix, Ep: epithelial layer, GC: ghost cells, MC: mesenchymal cells, SC: suprabasal fusiform and stellate cells, St: stromal layer. Bar: A: 100 lm; B: 50 lm. J Oral Pathol Med
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Table 3 Immunostaining and cellular localization of positive cells in neoplastic odontogenic epithelium of Calcifying Cystic Odontogenic Tumours
Basal layer Suprabasal fusiform and stellate cells Ghost cells
MMP-2
MMP-7
a
+ +a
+ +a
+
+
MMP-9
MMP-14
TIMP-2
+ +a,b
a
a,b
+
+
TIMP-3
TIMP-4
RECK
EMMPRIN
+ +a
+ +a,c
+ +a,b
+a,b +a,b
+
+
+
+
a
+
a,c
a,b
( ) no stain, (+) positive. a Cytoplasmic. b Cell membrane. c Nuclear.
r = 0.661), showing that when EMMPRIN expression decreases, stromal RECK expression also decreases.
Discussion We presented in this study the distribution of MMPs, TIMPs, RECK and EMMPRIN in both neoplastic odontogenic epithelium and adjacent stroma of CCOT. To our knowledge, this is the first report of TIMP-3, TIMP-4, RECK and EMMPRIN in this tumour and, because CCOT is a rare odontogenic lesion, the results obtained from the 18 samples used here represent robust information on the expression and distribution of these markers within the tumour. Traditionally, MMP biological functions have been associated with degradation and turnover of most ECM components. This functional simplistic conception has been used through the years to explain MMP involvement in embryonic development, homeostasis and disease. For instance, this has lead to the employment of MMP inhibitors in clinical trials for cancer treatment without success, raising the question of whether they can be really therapeutic targets. Recent studies using degradomics and proteomics approaches in MMP knockout mice models have changed MMP function dogma revealing dubious functions in tissues, both protective and destructive depending on the biological process involved (6–10). All MMPs evaluated in the present study were detected both in neoplastic odontogenic epithelium and adjacent stroma of CCOT in variable amounts, confirming previous findings (21–24). Although MMPs expression level in both neoplastic odontogenic epithelium and adjacent stroma of CCOT is known to be lower than in ameloblastoma (21, 25) and keratocystic odontogenic tumour (22, 25), our results revealed a higher expression ratio of MMP-9 in neoplastic epithelium compared to stroma in CCOT, mainly in ghost cells as already reported by others (25, 26). TIMPs have comparable abilities to inhibit the active forms of the MMPs and have shown to form tight complexes with active MMPs in a 1:1 stoichiometry. TIMPs inhibit all MMPs, but TIMP-1 is a poor inhibitor of MMP-14, MMP-17, MMP-19 and MMP-24. In addition to inhibiting MMP activity, TIMP-2 unique feature is to selectively interact with MMP-14 to facilitate the cell surface activation of pro-MMP-2 (tertiary complex TIMP-2/ MMP-14/pro-MMP-2). TIMPs show tissue-specific, constitutive or inducible expression, which is regulated at the transcriptional level by various cytokines and growth J Oral Pathol Med
factors. The roles of TIMPs in both normal physiology and in pathological processes have been investigated, and these have indicated that there is some functional redundancy. Elevated MMP/TIMP expression ratios have been associated with many diseases, while elevated MMP/TIMP protein level ratios are often viewed and interpreted with respect to the net increase in proteolytic activity of MMPs and disease pathology (27, 28). All TIMPs evaluated here were detected in both neoplastic odontogenic epithelium and adjacent stroma also in variable amounts, confirming previous findings of others (22, 24), with the exception of TIMP-3 and TIMP-4, which are described for the first time in this study. Among TIMPs, TIMP-3 is a glycoprotein that has complex sugar chains and is unique on interacting strongly with the ECM components (sulphated glycosaminoglycans) (28). Timp-3 gene deletion causes lung emphysema-like alveolar damage (29) and faster apoptosis of mammary epithelial cells after weaning (30). TIMP-3 has conflicting activities according to cell type; in some cells, it appears to promote the development of a transformed phenotype, promoting apoptosis in several tumour cell lines and in smooth muscle cells, but this appears to involve the modulation of MMPs activities. This suggests that TIMP-3 is a major regulator of MMP activities in vivo. Furthermore, TIMP-3 plays a key role in innate immunity by regulating the processing of tumour necrosis factor-a (TNF-a) by ADAM17 (31) is implicated in vascular inhibition by blockage of VEGF binding to its receptor (32), by interaction to angiotensin II type 2 (33), as well as TIMP-3 knockout mice have been showed neovascularization (34). Although TIMP-3 was strongly detected in both neoplastic odontogenic epithelium and adjacent stroma of CCOT, nothing is known in the literature about its expression in odontogenic tumours. Recent study demonstrated that TGF-b/Smad signalling in CCOT and adenomatoid odontogenic tumour is higher than in ameloblastoma, suggesting low and high neoplastic dynamics in these tumours, respectively (35). In our study, we could speculate that TIMP-3 might be expressed by TGF-b induction and act on the inhibition of MMP-2, MMP-7, MMP-9 and MMP-14 to prevent excess ECM degradation and avoid tumour expansion and progression. Human TIMP-4 is a non-glycosylated protein more closely related to TIMP-2 and -3 than to TIMP-1, binds to pro-MMP-2, and it is also a strong inhibitor of MMP-2 and MMP-14 (36); however, it is unable to promote the
0.004 50 ≤ 75%
> 75%
Neoplastic odontogenic epithelium vs. stroma P-value Percentage of neoplastic odontogenic epithelium immunostain Neoplastic odontogenic epithelium Stroma
Table 4 Semi-quantitative analysis of immunostaining positivity, intensity and percentual distribution within neoplastic odontogenic epithelium and adjacent stroma of Calcifying Cystic Odontogenic Tumours
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activation of the pro-MMP-2 (37). TIMP-4 presents high affinity to MMP-26 than to other MMPs (MMP26 > MMP-9 > MMP-7 > MMP-3 > MMP-2 > MMP-1, respectively) (38–40). Indeed, MMP-26 is expressed in CCOT (23), suggesting that TIMP-4 high expression observed here may be explained by the inhibition of ECM degradation by MMPs. RECK gene was first identified in cells transformed by the v-Ki-RAS oncogene that gave rise to flat colonies (morphological reversion) and exhibited MMP inhibition property (16). RECK mRNA is expressed ubiquitously in a wide range of normal tissues and untransformed cells, but is undetectable in tumour-derived cell lines and oncogenically transformed cells (41). RECK is a glycoprotein anchored on the cell membrane that specifically inhibits MMP-2 (16, 42–44), MMP-9 (16), MMP-14 (42, 43) and, apparently, MMP-7 (45), in vitro and in vivo. RECK is essential for normal embryonic and skeletal muscle development as well as cartilage differentiation, playing a role as a mediator of tissue remodelling and stabilization of tissue architecture, as shown in RECK null mice that die in utero with several connective tissue disruption. Interestingly, while RECK is required for angiogenesis in mouse embryos, it prevents angiogenesis in the tumour xenograft model (16, 42, 46, 47). In CCOT, RECK expression was more prominent in neoplastic odontogenic epithelium than in adjacent stroma. Concerning odontogenic tumours, RECK presents low expression in both ameloblastomas and malignant ameloblastic tumours mainly in neoplastic epithelium, suggesting that RECK contributes to tissue structuring and cell differentiation of the odontogenic epithelial tumours (48). We suggest here that an imbalance of MMP/RECK ratio contributes to changes in ECM composition of CCOT. EMMPRIN is a glycolized transmembrane protein known for its ability to induce MMP production in fibroblasts (49, 50). Elevated EMMPRIN levels have been found in several inflammatory and neoplastic conditions as well as in several tumours, including oral cavity tumours, being correlated with the aggressive and destructive nature of these conditions (51–53). In relation to odontogenic tumours, EMMPRIN was already detected in neoplastic epithelium of ameloblastomas and malignant ameloblastic tumours (48) and also in basal and suprabasal layers of odontogenic cysts (keratocyst odontogenic tumour, dentigerous cyst and periapical cyst) (54). In the present study, EMMPRIN positive cells were also mainly detected in the basal layer of the neoplastic odontogenic epithelium of CCOT, while weak staining in adjacent stroma was observed in a few cases. Moreover, the only correlation found was between stromal EMMPRIN and stromal RECK expression, revealing that when EMMPRIN expression decreases, stromal RECK also decreases. Taken together, our results revealed that TIMPs may inhibit MMPs in both CCOT tissue compartments; higher MMP expression is found in adjacent stroma in comparison with RECK and EMMPRIN; stromal MMPs expression is probably stimulated by epithelial EMMPRIN expression; and both stromal EMMPRIN expression and RECK expression decrease in a positive correlation manner.
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Table 5 Immunostaining and cellular localization of positive cells within the adjacent stroma of Calcifying Cystic Odontogenic Tumours
Mesenchymal cells Capillary vessels Extracellular matrix
MMP-2
MMP-7
MMP-9
MMP-14
TIMP-2
TIMP-3
TIMP-4
+ +a +
+ +a +
+
+ +b
+
+ +a +
+ +a,c +
a
a
a
b
a
a
a,c
RECK +
a,b
EMMPRIN +a,b
( ) no stain, (+) positive. a Cytoplasmic. b Cell membrane. c Nuclear.
Conclusion We conclude that these proteins/enzymes are differentially expressed in both neoplastic odontogenic epithelium and adjacent stroma of CCOT and suggest that they may participate on the behaviour of this odontogenic tumour through MMPs modulation by TIMPs, RECK and EMMPRIN.
14. 15. 16.
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43. Sasahara RM, Brochado SM, Takahashi C, et al. Transcriptional control of the RECK metastasis/angiogenesis suppressor gene. Cancer Detect Prev 2002; 26: 435–43. 44. Takagi S, Simizu S, Osada H. RECK negatively regulates matrix metalloproteinase-9 transcription. Cancer Res 2009; 69: 1502–8. 45. Omura A, Matsuzaki T, Mio K, et al. RECK forms cowbellshaped dimers and inhibits matrix metalloproteinase-catalyzed cleavage of fibronectin. J Biol Chem 2009; 284: 3461–9. 46. Echizenya M, Kondo S, Takahashi R, et al. The membraneanchored MMP-regulator RECK is a target of myogenic regulatory factors. Oncogene 2005; 24: 5850–7. 47. Welm B, Mott J, Werb Z. Developmental biology: vasculogenesis is a wreck without RECK. Curr Biol 2002; 12: R209– 11. 48. Kumamoto H, Ooya K. Immunohistochemical detection of MT1-MMP, RECK, and EMMPRIN in ameloblastic tumors. J Oral Pathol Med 2006; 35: 345–51. 49. Biswas C, Zhang Y, DeCastro R, et al. The human tumor cellderived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily. Cancer Res 1995; 55: 434–9. 50. Guo H, Zucker S, Gordon MK, Toole BP, Biswas C. Stimulation of matrix metalloproteinase production by recombinant extracellular matrix metalloproteinase inducer from transfected Chinese hamster ovary cells. J Biol Chem 1997; 272: 24–7. 51. Kanekura T, Chen X. CD147/basigin promotes progression of malignant melanoma and other cancers. J Dermatol Sci 2010; 57: 149–54. 52. Richard V, Pillai MR. The stem cell code in oral epithelial tumorigenesis: ‘the cancer stem cell shift hypothesis’. Biochim Biophys Acta 2010; 1806: 146–62. 53. Weidle UH, Scheuer W, Eggle D, Klostermann S, Stockinger H. Cancer-related issues of CD147. Cancer Genomics Proteomics 2010; 7: 157–69. 54. Ali MA. Expression of extracellular matrix metalloproteinase inducer in odontogenic cysts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106: 258–63.
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Acknowledgements The authors express special thanks to Elisa dos Santos and Juvani Lago (Department of Oral Pathology, Dental School, University of S~ao Paulo) for the histotechnical processing of the tissue samples.
J Oral Pathol Med
J Oral Pathol Med (2014) 43: 545–553 © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd wileyonlinelibrary.com/journal/jop
Calcifying Cystic Odontogenic Tumour: immunohistochemical expression of matrix metalloproteinases, their inhibitors (TIMPs and RECK) and inducer (EMMPRIN) F abio C. Prosd ocimi1, Camila O. Rodini2, Mari C. Sogayar3, Suzana C. O. M. Sousa1, Flavia C. A. Xavier4, Kati ucia B. S. Paiva1,5 1
Department of Oral Pathology, Dental School, University of S~ ao Paulo, S~ ao Paulo, Brazil; 2Laboratory of Histology, Department of Biological Sciences, Bauru Dental School, University of S~ ao Paulo Bauru, S~ ao Paulo, Brazil; 3Cell and Molecular Therapy Center (NUCEL), Department of Biochemistry, Chemistry Institute, University of S~ ao Paulo, S~ ao Paulo, Brazil; 4Department of Propedeutics and 5 Integrated Clinic, Dental School, Federal University of Bahia, Salvador, Brazil; Extracellular Matrix Biology and Cellular Interaction Group, Laboratory of Molecular Mechanisms of Cytoprotection, Department of Biochemistry, Chemistry Institute, University of S~ ao Paulo, S~ ao Paulo, Brazil
BACKGROUND: Calcifying cyst odontogenic tumour (CCOT) is a rare benign cystic neoplasm of odontogenic origin. MMPs are responsible for extracellular matrix remodelling and, together their inhibitors and inducer, determinate the level of its turnover in pathological processes, leading to an auspicious microenvironment for tumour development. Thus, our goal was to evaluate matrix metalloproteinases (MMPs-2, -7, -9 and -14), their inhibitors (TIMPs-2, -3, -4 and RECK) and its inductor (EMMPRIN) expression in CCOT. MATERIALS AND METHODS: We used 18 cases of CCOT submitted to immunolocalization of the target proteins and analysed in both neoplastic odontogenic epithelial and stromal compartments. RESULTS: All molecules evaluated were expressed in both compartments in CCOT. In epithelial layer, immunostaining for MMPs, TIMPs, RECK and EMMPRIN was found in basal, suprabasal spindle and stellate cells surrounding ghost cells and ghost cells themselves, except for MMP-9 and TIMP-2 which were only expressed by ghost cells. In stromal compartment, extracellular matrix, mesenchymal (MC) and endothelial cells (EC) were positive for MMP-2, -7, TIMP-3 and -4, while MMP-9, TIMP-2 and RECK were positive only in MC and MMP-14 only in EC. Statistical significance difference was found between both compartments for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001), being more expressed in epithelium than in stroma. Positive correlation between both stromal EMMPRIN and RECK expression was found (R = 0.661, P = 0.003).
Correspondence: Kati ucia B. S. Paiva, Department of Oral Pathology, Dental School, University of S~ao Paulo, Avenida Lineu Prestes, 748 bloco 9 superior, sala 959/974/976, Cidade Universitaria, S~ao Paulo, SP 05508000, Brazil. Tel/Fax: +551130913820, E-mail: [email protected] Accepted for publication December 21, 2013
CONCLUSIONS: We concluded that these proteins/ enzymes are differentially expressed in both epithelium and stroma of CCOT, suggesting an imbalance between MMPs and their inducer/inhibitors may contribute on the tumour behaviour. J Oral Pathol Med (2014) 43: 545–553 Keywords: Calcifying Cystic Odontogenic Tumour; EMMPRIN; matrix metalloproteinases; RECK; tissue inhibitors of MMPs
Introduction After the first description of the currently named calcifying cystic odontogenic tumour (CCOT) as a benign odontogenic cyst in 1962 by Gorlin and colleagues (1), the question on the cystic or neoplastic nature of this lesion raised several mode of classifications. Then, considering the different histomorphological and clinicopathological features of this set of tumours, the World Health Organization (WHO) in 2005 renamed these lesions as tumours exhibiting three variants (2): (I) calcifying cystic odontogenic tumour (CCOT) defined as a benign cystic neoplasm characterized by an ameloblastoma-like epithelium with ghost cells that may calcify; (II) dentinogenic ghost cell tumour (DGCT) defined as a locally invasive neoplasm characterized by ameloblastoma-like islands of epithelial cells in a mature connective tissue stroma; and (III) ghost cell odontogenic carcinoma (GCOC) defined as an uncommon malignant neoplasm that exhibits prominent mitotic activity, nuclear atypia and cellular pleomorphism, groups of ghost epithelial cells, necrosis, sometimes scarce mineralized or osteodentine-like (‘dentinoid’) material and infiltrative growth
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Table 1 Primary antibodies applied for immunohistochemistry Antibody
Clonality
Specificity
Source
Cataloge
MMP-2 MMP-7 MMP-9 MMP-14 TIMP-2 TIMP-3 TIMP-4 RECK EMMPRIN
Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Rabbit monoclonal Mouse monoclonal Rabbit polyclonal Rabbit polyclonal Rabbit monoclonal Rabbit polyclonal
Pro- and active forms Pro-form Pro-form Active form C-terminal region C-terminal region C-terminal region Total protein C-terminal region
Abcam Novus Biologicals Abcam Novus Biologicals Calbiochem Abcam Abcam Cell signalling Abcam
ab37150 NB600-1153 ab76003 NB110-57216 IM56 ab39185 ab58425 3433 ab70062
pattern (3). Thus, CCOT was classified as an epithelial odontogenic tumour with a contribution from odontogenic ectomesenchyme, showing histological evidence of an inductive change (4). Matrix metalloproteinases (MMPs) are the most important enzyme family responsible collectively for cleavage and degradation of all components of the extracellular matrix (ECM) and then generating bioactive molecules. Several pericellular and extracellular proteins are degraded or processed by MMPs (5). Nowadays, the proteolytic target spectrum of MMPs was amplified, including other molecules on cell membrane and pericellular proteins non-related to ECM (other proteinases, intracellular substrates, latent growth factors, proteinase inhibitors, chemostatic molecules, growth factor binding proteins, cell membrane receptor, cell–cell and ECM–cell adhesion molecules), leading to the regulation of cell behaviour in several pathways, mainly signal transduction (6–10). Mammalian MMPs (24 members) are classified in soluble and cell membrane anchor-MMP (MT-MMP) in relation to enzyme-substrate in vitro specificity and molecular structure, as well as discovery chronology. MMPs are divided in collagenases (MMP-1, -8 and -13), gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), matrilysins (MMP-7 and -26), MT-MMPs (MMP-14, -15, -16, 1-7, -24 and -25) and heterogeneous group (MMP-12, -19, -20, -21, -23, 27 and -28). Mostly, soluble MMPs are secreted as pro-enzymes, and activation is requested into ECM (11), whereas MT-MMPs are activated in intracellular space and expressed on cell membrane as active form (12). MMPs are regulated at transcriptional and post-transcriptional levels by their inhibitors and inductors, as well as interaction with specific ECM components (13). Gene expression is controlled by several stimulant and suppressor factors that influence many signalling pathways, such as integrins, ECM proteins, hormones, growth factors, oncogenes, cytokines, epigenetic regulation, cell stress, morphological changes and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) (14, 15). Regarding enzymatic inhibition, pro-MMP and its active form can be inhibited in the ECM by their inhibitors anchored on the cell membrane as reversion-inducing cysteine-rich protein with Kazal motifs (RECK) (16) and secreted into the ECM as tissue inhibitor of MMPs (TIMPs) (17). Most MMPs play central role in physiological conditions (stem cell differentiation, proliferation, cell mobility, tissue remodelling, wound J Oral Pathol Med
Dilution 1:250 1:500 1:200 1:500 1:500 1:200 1:200 1:50 1:500
healing, angiogenesis and apoptosis), and an imbalance between MMPs, their inhibitors and inducer is implicated in different pathological processes (cancer, degenerative diseases) through several mechanisms, mainly related to tissue destruction, fibrosis and changes in the microenvironment (ECM composition). MMP overexpression has already been correlated with tumoural aggressiveness, stage and prognosis in a large range of malignant tumours (18–20); however, little is known about ECM changes and cell–ECM interactions in odontogenic tumours. Thus, our aim was to evaluate the immunoexpression of some MMPs (-2, -7, -9 and -14), TIMPs (-2, -3 and -4), RECK and EMMPRIN in epithelial and stromal compartments of CCOT.
Material and methods Patient tissue samples The research was approved by the Ethics Committee of the Dental School, University of S~ao Paulo. Eighteen cases of calcifying cystic odontogenic tumour (CCOT), intraosseous and without other associated odontogenic tumours were obtained from the files of the Pathology Laboratory, Department of Stomatology (from 2000 to 2012). Histopathological diagnosis was confirmed by an oral pathologist
Table 2 Main clinical data of Calcifying Cystic Odontogenic Tumour cases in the studied sample Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Gender Male Male Male Female Female Male Female Male Female Female Female Male Female Female Male Male Male Male
Age (years)
Site
39 40 36 21 27 28 26 19 14 20 20 26 28 21 47 39 15 25
Maxilla Maxilla Maxilla Maxilla Mandible Uninformed Anterior maxilla Anterior maxilla Right maxilla Anterior maxilla Anterior maxilla Right angle mandible Uninformed Maxilla Mandible Mandible Right maxilla Anterior maxilla
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(S.O.M.S.) through the slides review stained with haematoxylin and eosin, according to the WHO classification of head and neck tumours (3). The main clinical data as gender, age and tumour site were also collected.
3% H2O2 solution for 45 min, and the free charges were neutralized by 1% borax solution for 15 min. Heat-induced epitope retrieval (HIER) was performed with phosphatecitrate buffer – 93–96°C, pH 6.0 for 20 min (P4809; SigmaAldrich, St. Louis, MO, USA – except for MMP-2, for which no epitope retrieval was recommended by the manufacturer), and non-specific binding was blocked by Protein Block Serum-Free (X0909; DAKO, Carpinteria, CA, USA) for 10 min. The sections were incubated with primary antibodies (Table 1) in a humid chamber at 4°C
Immunohistochemistry Formalin-fixed paraffin-embedded (FFPE) CCOT tissue sections (4 lm thickness) were mounted on silane-coated slides. After deparaffinization and rehydration in ethanolgraded series, the endogenous peroxidase was blocked by A
B
C
D
E
F
G
H
547
Figure 1 Immunoexpression of matrix metalloproteinases 2, 7, 9 and 14 in calcifying cystic odontogenic tumour. Bl: basal layer, CV: capillary vessels, ECM: extracellular matrix, Ep: epithelial layer, IC: inflammatory cells, GC: ghost cells, MC: mesenchymal cells, SC: suprabasal spindle and stellate cells, St: stromal layer. Bar: 50 and 200 lm. J Oral Pathol Med
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overnight, and detection was performed using EnVisionTM + Dual Link System-HRP (K4061; DAKO) or LSAB + System-HRP (K0690; DAKO). The immunostaining was revealed by Liquid DAB+Substrate-Chromogen System (K3468; DAKO), and the sections were counterstained with Mayer′s haematoxylin. All rinses were performed using 0.1% Triton X-100/PBS. Antibody diluent with background reducing components (S3022; DAKO) was used for both primary antibody dilution and negative control (primary antibody was replaced by this solution). Human oral
squamous cell carcinoma biopsy was used as positive control for all antibodies. Interpretation of immunohistochemical staining The immunohistochemical analysis was performed by two trained examiners (F.C.A.X. and C.O.R.) at different times. Semi-quantitative analysis of immunostained cells in both neoplastic odontogenic epithelium and adjacent stroma was performed according to immunostaining intensity: no staining ( ), weak (+), moderate (++) and intense (+++). The A
B
C
D
E
F
G
H
Figure 2 Immunoexpression of tissue inhibitors of matrix metalloproteinases (TIMPs 2, 3 and 4) and RECK in calcifying cystic odontogenic tumour. Bl: basal layer, CV: capillary vessels, ECM: extracellular matrix, Ep: epithelial layer, GC: ghost cells, MC: mesenchymal cells, SC: suprabasal spindle and stellate cells, St: stromal layer. Bar: A: 100 lm; B, D, F, H: 50 lm; C, E, G: 200 lm. J Oral Pathol Med
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percentage of immunopositive cells restricted to the neoplastic odontogenic epithelium was estimated in 2009 microscopic magnification and scored as 1 (≤ 10% of stained epithelial cells), 2 (> 10 ≤ 50%), 3 (> 50 ≤ 75%) and 4 (> 75%). Immunostaining distribution was described within epithelium (basal layer, suprabasal fusiform, stellate and ghost cells) and within stroma (mesenchymal and endothelial cells and extracellular matrix) and regarding cellular compartment as cell membrane, cytoplasmic and/or nuclear for all molecules. Statistical analysis Statistical analysis was accomplished using the statistical package Statistical Package for Social Sciences (SPSS), version 20.0 (IBM Corp., Armonk, NY, USA). Comparison of absence (negative cases – no detected staining) or presence (all positive cases independently of immunostaining intensity) found for each protein assessed between tissue compartments (neoplastic odontogenic epithelium or adjacent stroma) was performed using Fisher’s exact test, while the correlation between proteins expression within tissue compartments was performed using Spearman’s rank correlation coefficient test. For both tests, P-value < 0.05 was considered to indicate statistical significance.
Results Clinical data demonstrated that the gender distribution of CCOT was almost equal between males and females (10:8), and the individuals were usually young to middle-aged (age mean 27.7, SD = 9.36). The maxilla, predominantly the anterior region, was in fact the most affected site (Table 2). Immunohistochemical evaluation showed that all enzymes/proteins were expressed in variable amounts in both neoplastic odontogenic epithelium and adjacent stroma in all cases of CCOT. Regarding epithelial immunostaining, wide positivity for MMP-2 (n = 18/18) (Fig. 1A, B), MMP7 (n = 18/18) (Fig. 1C, D), MMP-14 (n = 18/18) (Fig. 1G, H), TIMP-3 (n = 18/18) (Fig. 2C, D), TIMP-4 (n = 18/18) (Fig. 2E, F), RECK (n = 14/18) (Fig. 2G, H) and EMMPRIN (n = 14/18) (Fig. 3A, B) was found in basal, spindle suprabasal and stellate epithelial cells surrounding ghost cells as well as ghost cells themselves. MMP-9 (n = 16/18)
A
(Fig. 1C, D) and TIMP-2 (n = 15/18) (Fig. 2A, B) were only seen in ghost cells (Table 3). Cytoplasmic immunostaining alone was found for MMP-2, MMP-7 and TIMP-3, while simultaneous cytoplasmic and cell membrane immunostaining were found for MMP-14, RECK and EMMPRIN. Only TIMP-4 showed both cytoplasmic and nuclear immunostaining patterns (Table 4). Concerning the percentage of neoplastic epithelial immunopositive cells in CCOT, MMP-2, MMP-7, MMP-14 and TIMP-3 presented score 3, while TIMP-4 showed score 4. On the other hand, scarce immunostaining corresponding to score 2 was observed for MMP-9, TIMP-2, RECK and EMMPRIN in most cases (Table 4). Regarding immunostaining intensity, cells were intense or moderately stained for MMP-2 (n = 18/18), MMP-7 (n = 12/18), MMP-9 (n = 11/18), MMP-14 (n = 13/18), TIMP-3 (n = 16/18) and TIMP-4 (n = 18/18), whereas weak staining was observed for TIMP-2 (n = 13/18), RECK (n = 11/18) and EMMPRIN (n = 8/18) in most cases (Table 4). When adjacent stroma was analysed, both mesenchymal and endothelial cells, as well as the ECM, were also immunopositive for MMP-2, MMP-7, TIMP-3, TIMP-4. MMP-9, TIMP-2, while positivity for RECK was only seen within mesenchymal cells, and MMP-14 was restricted to endothelial cells (Table 5). Cytoplasmic immunostaining alone was detected for MMP-2, MMP-7, MMP-9 and TIMP-2, and cell membrane immunostaining alone was seen for MMP-14. Simultaneous cytoplasmic and nuclear immunostaining were found for TIMP-4, and both cytoplasmic and cell membrane immunostaining were found for RECK and EMMPRIN. Regarding immunostaining intensity, intense or moderate staining was observed for MMP-2, MMP-7, MMP-14, TIMP-2, TIMP-3 and TIMP-4, whereas weak staining for MMP-9 was observed in most cases. For RECK and EMMPRIN, only a few cases presented weak positivity (n = 3 and n = 2, respectively) (Tables 3 and 5). Comparing the immunostaining positivity for each protein between tissue compartments (neoplastic odontogenic epithelium and adjacent stroma), a statistically significant positivity for MMP-9 (P < 0.001), RECK (P = 0.004) and EMMPRIN (P < 0.001) was found within the neoplastic odontogenic epithelium (Table 4). Furthermore, strong positive correlation between stromal EMMPRIN and stromal RECK expression was found (P = 0.003 and
549
B
Figure 3 Immunoexpression of inducer of matrix metalloproteinases (EMMPRIN) in calcifying cystic odontogenic tumour. Bl: basal layer, CV: capillary vessels, ECM: extracellular matrix, Ep: epithelial layer, GC: ghost cells, MC: mesenchymal cells, SC: suprabasal fusiform and stellate cells, St: stromal layer. Bar: A: 100 lm; B: 50 lm. J Oral Pathol Med
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Table 3 Immunostaining and cellular localization of positive cells in neoplastic odontogenic epithelium of Calcifying Cystic Odontogenic Tumours
Basal layer Suprabasal fusiform and stellate cells Ghost cells
MMP-2
MMP-7
a
+ +a
+ +a
+
+
MMP-9
MMP-14
TIMP-2
+ +a,b
a
a,b
+
+
TIMP-3
TIMP-4
RECK
EMMPRIN
+ +a
+ +a,c
+ +a,b
+a,b +a,b
+
+
+
+
a
+
a,c
a,b
( ) no stain, (+) positive. a Cytoplasmic. b Cell membrane. c Nuclear.
r = 0.661), showing that when EMMPRIN expression decreases, stromal RECK expression also decreases.
Discussion We presented in this study the distribution of MMPs, TIMPs, RECK and EMMPRIN in both neoplastic odontogenic epithelium and adjacent stroma of CCOT. To our knowledge, this is the first report of TIMP-3, TIMP-4, RECK and EMMPRIN in this tumour and, because CCOT is a rare odontogenic lesion, the results obtained from the 18 samples used here represent robust information on the expression and distribution of these markers within the tumour. Traditionally, MMP biological functions have been associated with degradation and turnover of most ECM components. This functional simplistic conception has been used through the years to explain MMP involvement in embryonic development, homeostasis and disease. For instance, this has lead to the employment of MMP inhibitors in clinical trials for cancer treatment without success, raising the question of whether they can be really therapeutic targets. Recent studies using degradomics and proteomics approaches in MMP knockout mice models have changed MMP function dogma revealing dubious functions in tissues, both protective and destructive depending on the biological process involved (6–10). All MMPs evaluated in the present study were detected both in neoplastic odontogenic epithelium and adjacent stroma of CCOT in variable amounts, confirming previous findings (21–24). Although MMPs expression level in both neoplastic odontogenic epithelium and adjacent stroma of CCOT is known to be lower than in ameloblastoma (21, 25) and keratocystic odontogenic tumour (22, 25), our results revealed a higher expression ratio of MMP-9 in neoplastic epithelium compared to stroma in CCOT, mainly in ghost cells as already reported by others (25, 26). TIMPs have comparable abilities to inhibit the active forms of the MMPs and have shown to form tight complexes with active MMPs in a 1:1 stoichiometry. TIMPs inhibit all MMPs, but TIMP-1 is a poor inhibitor of MMP-14, MMP-17, MMP-19 and MMP-24. In addition to inhibiting MMP activity, TIMP-2 unique feature is to selectively interact with MMP-14 to facilitate the cell surface activation of pro-MMP-2 (tertiary complex TIMP-2/ MMP-14/pro-MMP-2). TIMPs show tissue-specific, constitutive or inducible expression, which is regulated at the transcriptional level by various cytokines and growth J Oral Pathol Med
factors. The roles of TIMPs in both normal physiology and in pathological processes have been investigated, and these have indicated that there is some functional redundancy. Elevated MMP/TIMP expression ratios have been associated with many diseases, while elevated MMP/TIMP protein level ratios are often viewed and interpreted with respect to the net increase in proteolytic activity of MMPs and disease pathology (27, 28). All TIMPs evaluated here were detected in both neoplastic odontogenic epithelium and adjacent stroma also in variable amounts, confirming previous findings of others (22, 24), with the exception of TIMP-3 and TIMP-4, which are described for the first time in this study. Among TIMPs, TIMP-3 is a glycoprotein that has complex sugar chains and is unique on interacting strongly with the ECM components (sulphated glycosaminoglycans) (28). Timp-3 gene deletion causes lung emphysema-like alveolar damage (29) and faster apoptosis of mammary epithelial cells after weaning (30). TIMP-3 has conflicting activities according to cell type; in some cells, it appears to promote the development of a transformed phenotype, promoting apoptosis in several tumour cell lines and in smooth muscle cells, but this appears to involve the modulation of MMPs activities. This suggests that TIMP-3 is a major regulator of MMP activities in vivo. Furthermore, TIMP-3 plays a key role in innate immunity by regulating the processing of tumour necrosis factor-a (TNF-a) by ADAM17 (31) is implicated in vascular inhibition by blockage of VEGF binding to its receptor (32), by interaction to angiotensin II type 2 (33), as well as TIMP-3 knockout mice have been showed neovascularization (34). Although TIMP-3 was strongly detected in both neoplastic odontogenic epithelium and adjacent stroma of CCOT, nothing is known in the literature about its expression in odontogenic tumours. Recent study demonstrated that TGF-b/Smad signalling in CCOT and adenomatoid odontogenic tumour is higher than in ameloblastoma, suggesting low and high neoplastic dynamics in these tumours, respectively (35). In our study, we could speculate that TIMP-3 might be expressed by TGF-b induction and act on the inhibition of MMP-2, MMP-7, MMP-9 and MMP-14 to prevent excess ECM degradation and avoid tumour expansion and progression. Human TIMP-4 is a non-glycosylated protein more closely related to TIMP-2 and -3 than to TIMP-1, binds to pro-MMP-2, and it is also a strong inhibitor of MMP-2 and MMP-14 (36); however, it is unable to promote the
0.004 50 ≤ 75%
> 75%
Neoplastic odontogenic epithelium vs. stroma P-value Percentage of neoplastic odontogenic epithelium immunostain Neoplastic odontogenic epithelium Stroma
Table 4 Semi-quantitative analysis of immunostaining positivity, intensity and percentual distribution within neoplastic odontogenic epithelium and adjacent stroma of Calcifying Cystic Odontogenic Tumours
MMPs, TIMPs, RECK and EMMPRIN in CCOT Prosd ocimi et al.
activation of the pro-MMP-2 (37). TIMP-4 presents high affinity to MMP-26 than to other MMPs (MMP26 > MMP-9 > MMP-7 > MMP-3 > MMP-2 > MMP-1, respectively) (38–40). Indeed, MMP-26 is expressed in CCOT (23), suggesting that TIMP-4 high expression observed here may be explained by the inhibition of ECM degradation by MMPs. RECK gene was first identified in cells transformed by the v-Ki-RAS oncogene that gave rise to flat colonies (morphological reversion) and exhibited MMP inhibition property (16). RECK mRNA is expressed ubiquitously in a wide range of normal tissues and untransformed cells, but is undetectable in tumour-derived cell lines and oncogenically transformed cells (41). RECK is a glycoprotein anchored on the cell membrane that specifically inhibits MMP-2 (16, 42–44), MMP-9 (16), MMP-14 (42, 43) and, apparently, MMP-7 (45), in vitro and in vivo. RECK is essential for normal embryonic and skeletal muscle development as well as cartilage differentiation, playing a role as a mediator of tissue remodelling and stabilization of tissue architecture, as shown in RECK null mice that die in utero with several connective tissue disruption. Interestingly, while RECK is required for angiogenesis in mouse embryos, it prevents angiogenesis in the tumour xenograft model (16, 42, 46, 47). In CCOT, RECK expression was more prominent in neoplastic odontogenic epithelium than in adjacent stroma. Concerning odontogenic tumours, RECK presents low expression in both ameloblastomas and malignant ameloblastic tumours mainly in neoplastic epithelium, suggesting that RECK contributes to tissue structuring and cell differentiation of the odontogenic epithelial tumours (48). We suggest here that an imbalance of MMP/RECK ratio contributes to changes in ECM composition of CCOT. EMMPRIN is a glycolized transmembrane protein known for its ability to induce MMP production in fibroblasts (49, 50). Elevated EMMPRIN levels have been found in several inflammatory and neoplastic conditions as well as in several tumours, including oral cavity tumours, being correlated with the aggressive and destructive nature of these conditions (51–53). In relation to odontogenic tumours, EMMPRIN was already detected in neoplastic epithelium of ameloblastomas and malignant ameloblastic tumours (48) and also in basal and suprabasal layers of odontogenic cysts (keratocyst odontogenic tumour, dentigerous cyst and periapical cyst) (54). In the present study, EMMPRIN positive cells were also mainly detected in the basal layer of the neoplastic odontogenic epithelium of CCOT, while weak staining in adjacent stroma was observed in a few cases. Moreover, the only correlation found was between stromal EMMPRIN and stromal RECK expression, revealing that when EMMPRIN expression decreases, stromal RECK also decreases. Taken together, our results revealed that TIMPs may inhibit MMPs in both CCOT tissue compartments; higher MMP expression is found in adjacent stroma in comparison with RECK and EMMPRIN; stromal MMPs expression is probably stimulated by epithelial EMMPRIN expression; and both stromal EMMPRIN expression and RECK expression decrease in a positive correlation manner.
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Table 5 Immunostaining and cellular localization of positive cells within the adjacent stroma of Calcifying Cystic Odontogenic Tumours
Mesenchymal cells Capillary vessels Extracellular matrix
MMP-2
MMP-7
MMP-9
MMP-14
TIMP-2
TIMP-3
TIMP-4
+ +a +
+ +a +
+
+ +b
+
+ +a +
+ +a,c +
a
a
a
b
a
a
a,c
RECK +
a,b
EMMPRIN +a,b
( ) no stain, (+) positive. a Cytoplasmic. b Cell membrane. c Nuclear.
Conclusion We conclude that these proteins/enzymes are differentially expressed in both neoplastic odontogenic epithelium and adjacent stroma of CCOT and suggest that they may participate on the behaviour of this odontogenic tumour through MMPs modulation by TIMPs, RECK and EMMPRIN.
14. 15. 16.
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Acknowledgements The authors express special thanks to Elisa dos Santos and Juvani Lago (Department of Oral Pathology, Dental School, University of S~ao Paulo) for the histotechnical processing of the tissue samples.
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