Functional role of matrix metalloproteinase-28 in the oral squamous cell carcinoma

doi:10.1016/j.oraloncology.2005.12.012

Oral Oncology

Volume 42, Issue 9, October 2006, Pages 907-913

https://doi.org/10.1016/j.oraloncology.2005.12.012 Get rights and content

Summary

The newly identified MMP-28 has been shown to be expressed in several types of carcinomas, however, its functional role in transformation events is unknown. This study was to assess whether this proteinase plays a role in oral tumor malignancy. By using RT-PCR, we found that expression of MMP-28 was significantly higher in 92 oral squamous cell carcinomas (OSCCs) (52/92, 56.5%) than in seven oral premalignant lesions (OPMLs) (0/7, 0%) (P = 0.004). No statistically significant correlation was found between MMP-28 expression and tumor stage, thickness, size, and metastasis. Both mRNA and protein of MMP-28 were preferentially concentrated in OSCC specimens than in neighboring tissues as analyzed by semi-quantitative RT-PCR (P = 0.015) and immunohistochemistry, respectively. Transfection of OSCC and esophageal carcinoma cell lines with MMP-28 antisense oligodeoxynucleotide (AODN) resulted in the reduced secretion of MMP-28 protein and the ability of colony formation in soft agar without affecting cell growth. Our findings show the close correlation between MMP-28 and OSCC, and support a role for MMP-28 in the anchorage-independent growth of both OSCC and esophageal carcinomas.

Introduction

Matrix metalloproteinases (MMPs) play a central role in many physiological functions, such as development, wound healing, inflammation, and angiogenesis.1, 2, 3, 4, 5 Over the past two decades, the relevance of the MMP family in the oncology has been considerably studied. These enzymes were associated with the invasive properties of tumor cells, owing to their ability to degrade all major protein components of the extracellular matrix (ECM) and basement membranes. Elevated levels of some certain MMPs can be detected in tumor tissues or sera of patients with advanced cancers. They can also serve as prognostic indicators in various cancers, such as the head and neck carcinomas.6, 7, 8

Inhibition of tumor growth, migration, invasion, and metastasis by specific MMP inhibitors or antisense strategies has been demonstrated in numerous studies. For instances, expression of MMP-9 antisense construct inhibited the growth and invasion of a human glioblastoma cell line.9, 10 A newly synthesized gelatinase inhibitor, ONO-4817, alone or combined with docetaxel, suppressed the metastatic potential of MMP-expressing lung carcinoma cell lines in immunodeficient mice.11, 12 Moreover, a cyclic peptide CTTHWGFTLC, selected from a phage display peptide libraries, demonstrated to specifically inhibit the activities of MMP-2 and MMP-9, blocked the migration of human endothelial and tumorous cells as well as to prevent tumor growth and invasion in animal models.¹³

Up-to-date, MMP-28 (epilysin), structurally belonging to the MMP-19 subfamily, may represent the newest MMP member and is expressed in a variety of normal and carcinoma tissues.14, 15 In wounded human skin, MMP-28 protein is prominently stained in basal keratinocytes both at and some distance from the wound edge. It was also present in fetal tissues and rhesus monkey placenta during early pregnancy.14, 16 Therefore, its function in normal tissue homeostasis, wound repair, and development, as well as in tumor progression is suggested.14, 15, 16 MMP-28 may also be endowed with immunological functions, as it was found in T lymphocytes from the blood of normal individual, and elevated in the cartilage from patients with osteoarthritis.17, 18 These findings further amplify the diversified functions of MMP-28.

The involvement of MMP-28 in pathological processes remains obscure. For examples, in the inflammatory conditions, it was shown to be upregulated in osteoarthritis by immunohistochemistry (IHC),¹⁷ but downregulated in inflammatory bowel disease or ischemic colitis by real-time PCR.¹⁹ Furthermore, MMP-28 was demonstrated to be widely expressed in a variety of carcinomas, such as pancreatic adenocarcinoma, ovarian carcinoma, and colon adenocarcinoma.¹⁴ On the other hand, this proteinase was shown to be downregulated in colon cancers, and its function in tissue homeostasis rather than in tumor progression was emphasized.¹⁹

To assess whether MMP-28 plays a role in oral tumors, we firstly examined the expression pattern of MMP-28 transcript in surgical specimens of seven oral premalignant lesions (OPMLs) and 92 oral squamous cell carcinomas (OSCCs) by RT-PCR. Distribution of MMP-28 protein was also analyzed by IHC in another 10 OSCC tissue sections. To explore its possible functions, an OSCC cell line, Meng-1 (OECM-1),²⁰ and an esophageal carcinoma cell line, CE81T/VGH,21, 22 were subjected to specific inhibition of MMP-28 by antisense oligodeoxynucleotide (AODN) transfection. Cell proliferation and anchorage-independent growth were subsequently analyzed.

Section snippets

Tumor specimens

Surgical specimens of seven oral premalignant lesions (OPMLs) and 92 oral squamous cell carcinomas (OSCCs) were collected from Chi-Mei Medical Center and preserved at −80 °C for subsequent RNA preparation. Twenty-four of the 92 OSCC specimens as well as their neighboring tissues were simultaneously collected for the comparison of MMP-28 mRNA level between the malignant and the surrounding tissues.

Semi-quantitative RT-PCR

Protocols of tissue homogenate, total cellular RNA preparation and RT-PCR conditions were described

Incidence of MMP-28 in OPMLs and OSCCs

By using the RT-PCR analysis, MMP-28 message was preferentially detected in OSCCs (52/92, 56.5%) rather than in OPMLs (0/7, 0%) (P = 0.004, Table 2). Twenty-four of the 52 MMP-28-positive OSCC samples (T1–T24) are shown in Figure 1A. On the other hand, MMP-28 incidence was not statistically correlated with tumor stage, thickness, size, and lymph node metastasis (Table 2).

Elevated transcription of MMP-28 in OSCCs

To compare the expression level of MMP-28 between OSCCs and their surrounding tissues, T1–T24 and their neighboring tissues

Discussion

According to our present study, MMP-28 message was preferentially expressed in OSCCs (52/92, 56.5%) than in those of OPMLs (0/7, 0%) (P = 0.004, Table 2), and was dominantly transcribed in OSCC specimens than in those of neighboring tissues (P = 0.015, Fig. 1). In consistent with the distribution of mRNA, MMP-28 protein was also mainly concentrated in OSCCs than in those of surrounding tissues (Fig. 2). These expression patterns suggested that MMP-28 might play an important role in OSCC. It is

Acknowledgement

This work was supported in part by Chi-Mei Medical Center (CMFHR 9340 and CMFHR 9425) and National Science Council (NSC 92-2314-B-366-001 and NSC 92-2626-B-041-006). The first two and the corresponding authors contributed equally to this work. We would also like to thank Mr. Tai-Chi Chen, Ms. Yu-Rong Shu, and Ms. Ya-Yun Shi for their technical assistance.

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Citation Excerpt :
Only in the case of colon carcinoma MMP-28, unlike other MMPs, is downregulated, as compared to normal tissues (Bister et al., 2004). Lin and colleagues have observed a closed correlation between MMP-28 levels and oral squamous cell carcinoma (OSCC), supporting a role for MMP-28 in the anchorage-independent growth of both OSCC and esophageal carcinomas (Lin et al., 2006). Importantly, an overexpression of MMP-28 in lung adenocarcinoma cells induces an epithelial to mesenchymal transition (EMT) via activation of the latent transforming growth factor β (TGF-β) (Illman et al., 2006; Illman et al., 2008).
Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn²⁺ atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes.
Chronic exposure to cigarette smoke increases matrix metalloproteinases and Filaggrin mRNA expression in oral keratinocytes: Role of nicotine stimulation
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MMP-28 is normally expressed in the basal and suprabasal epidermis in intact skin, whilst in injured skin, its expression is seen in basal keratinocytes during wound healing18,21 and in hypertrophic scars.22 It has been observed that MMP-28 expression in primary keratinocytes is induced by tumour necrosis factor α (TNFα) and it is increased in oral squamous cell carcinomas (OSCC).23 Moreover MMP-28 can induce an epithelial to mesenchymal transition (EMT) via activation of latent TGFβ.11,12
The vegetal alkaloid nicotine has been proved to modify the expression of many keratinocyte markers. In this study, the basal expression of MMP-2, MMP-9, MMP-28, and Filaggrin has been evaluated in oral keratinocytes, in order to collect information about the ability of cigarette smoke to modify the basal expression pattern of these key enzymes in the absence of evident clinical signs in the oral epithelium. MMP-2, MMP-9, MMP-28, and Filaggrin basal expression was investigated by RT-PCR in oral keratinocytes derived from smokers (n = 11), non-smokers (n = 11), and ex-smokers (n = 6) healthy volunteers. Moreover keratinocytes from non-smokers volunteers were stimulated in vitro by a single dose administration of nicotine (10 μM) in order to estimate the effect of nicotinic receptors activation on the basal expression of the studied markers. RT-PCR analysis showed that all the markers studied were overexpressed in keratinocytes from smoker donors compared to control keratinocytes, while a single dose of nicotine was able to induce only Filaggrin expression in keratinocytes from non-smoking donors. Markers expression in ex-smoker donors was similar to that observed in normal non-smoker donors. These data indicate for the first time that cigarette smoking affects basal expression of some important markers in oral mucosa keratinocytes in vivo in the absence of clinical signs and that smoke quitting restores basal expression levels of these markers.
Expression and function of matrix metalloproteinase (MMP)-28
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Matrix metalloproteinase-28 (MMP-28, epilysin) is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues. In epithelial cells, over-expression of MMP-28 mediates irreversible epithelial to mesenchymal transition concomitant with loss of E-cadherin from the cell surface and an increase in active transforming growth factor beta. We recently reported the expression of MMP-28 in both cartilage and synovium where expression is increased in patients with osteoarthritis.
In human chondrosarcoma cells MMP-28 was activated by proprotein convertases and the active form of the enzyme preferentially associated with the extracellular matrix in a C-terminal independent manner. over-expression of MMP-28 in chondrosarcoma cells led to altered cell morphology with increased organisation of actin. Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased. MMP-28 was localised to the cell surface, at least transiently, in a C-terminal dependent manner. Heparin prevented both extracellular matrix association and cell surface binding of MMP-28 suggesting that both are via heparan sulphate proteoglycans. Over-expression of activatable MMP-28, but not catalytically inactive EA mutant increased the expression and activity of MMP-2, and all forms of MMP-28 tested increased expression of MMP19 and TIMP3 mRNA.
These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour. Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.
Epilysin (MMP-28) is deposited to the basolateral extracellular matrix of epithelial cells
2009, Matrix Biology
Epilysin (MMP-28) is a conserved member of the matrix metalloproteinase (MMP) family. It is expressed in various normal tissues, and induced in wounds and in developing and regenerating nerves. Epilysin induces TGF-β mediated epithelial to mesenchymal transition, but its other functions are largely unknown. We have characterized the localization of both catalytically active and mutated inactive, overexpressed epilysin in established epithelial cell lines. We found that epilysin was localized abundantly to the basolateral side of the cells and associated with the extracellular matrix (ECM) as verified by immunoblotting and confocal microscopy. Overexpression of epilysin in MDCK cells resulted in a drastic reduction of basolateral ECM, as observed by the disappearance of collagen type IV, laminin and fibronectin. Cultivation of epilysin expressing MDCK cells in defined serum free medium resulted in the restoration of these proteins to the ECM. The levels of fibronectin and collagen IV were, however, reduced in epilysin expressing cells under the serum free conditions, and degradation fragments of collagen IV were detected supporting the activation of proteolysis by epilysin. Epilysin was observed in its unprocessed 50 kDa active form in the ECM of MDCK cells under serum free conditions whereas in cells cultured in serum containing it was processed to the 48 kDa form. Current results indicate that epilysin associates with the basolateral ECM of cultured epithelial cells, where it plausibly plays a role in the regulation of matrix composition and turnover.
Upregulation of matrix metalloproteinase-1 (MMP-1) expression in oral carcinomas of betel quid (BQ) users: Roles of BQ ingredients in the acceleration of tumour cell motility through MMP-1
2008, Archives of Oral Biology
Citation Excerpt :
The sites, stages, and differentiation states of these specimens were listed in Table 1. The protocols of RNA isolation and reverse transcription, as well as the PCR conditions were described previously.14 PCR primers of MMP-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes and the expected sizes of PCR products are listed in Table 2.
Matrix metalloproteinases (MMPs) are commonly expressed in carcinomas including oral squamous cell carcinomas (OSCCs). On the other hand, some evidences suggested that ingredients of betel quid (BQ) inhibit the activity and/or expression of some MMPs thought to be the pathogenesis of oral submucous fibrosis. This study was to analyse whether MMP-1 expression is inhibited in OSCC specimens from BQ users and in cell lines survived from the challenge of BQ ingredients. We found that MMP-1 mRNA was expressed in all the tested 27 OSCC. Levels of MMP-1 mRNA and protein were significantly elevated in the tested five OSCC specimens than in their adjacent tissues (P < 0.001 and 0.05, respectively). Esophageal carcinoma (CE81T/VGH) and OSCC (OECM-1) cell lines survived from the cytotoxic BQ extract (BQE) and arecoline selection process were found to express higher MMP-1 mRNA and protein levels, or to exhibit a significant acceleration of two-dimensional (2D) motility than their non-selected parental cells. The enhanced motility was further demonstrated to be specifically and significantly inhibited by the MMP-1 neutralizing antibody and/or by the transfection of an MMP-1 specific antisense oligodeoxynucleotide. These results suggest that in some carcinomas of the upper aerodigestive tract, BQ usage may upregulate MMP-1 expression in the survived tumour cells, and increase their mobility in an MMP-1-dependent manner.

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Functional role of matrix metalloproteinase-28 in the oral squamous cell carcinoma

Summary

Introduction

Section snippets

Tumor specimens

Semi-quantitative RT-PCR

Incidence of MMP-28 in OPMLs and OSCCs

Elevated transcription of MMP-28 in OSCCs

Discussion

Acknowledgement

Gene

J Biol Chem

Exp Cell Res

J Biol Chem

J Biol Chem

Matrix metalloproteinase stromelysin-3 in development and pathogenesis

Histol Histopathol

Role of matrix metalloproteinases and their inhibition in cutaneous wound healing and allergic contact hypersensitivity

Ann N Y Acad Sci

Molecular mechanism for activation and regulation of matrix metalloproteinases during bacterial infections and respiratory inflammation

Biol Chem

Matrix metalloproteinases and angiogenesis

J Cell Mol Med

Metalloproteinases and their inhibitors in tumor angiogenesis

Int J Cancer

Matrix metalloproteinases: a review

Crit Rev Oral Biol Med

Expression of matrix metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 9 in carcinoma of the head and neck

Cancer

The role of matrix metalloproteinases in squamous cell carcinomas of the head and neck

Clin Exp Meta

Selective suppression of matrix metalloproteinase-9 in human glioblastoma cells by antisense gene transfer impairs glioblastoma cell invasion

Cancer Res

Adenovirus-mediated expression of antisense MMP-9 in glioma cells inhibits tumor growth and invasion

Oncogene

A third-generation matrix metalloproteinase (MMP) inhibitor (ONO-4817) combined with docetaxel suppresses progression of lung micrometastasis of MMP-expressing tumor cells in nude mice

Int J Cancer

Organ heterogeneity of host-derived matrix metalloproteinase expression and its involvement in multiple-organ metastasis by lung cancer cell lines

Cancer Res