Matrix Metalloproteinases Participate in Osteosarcoma Invasion

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Background

Osteosarcoma (OS) is a highly malignant bone tumor and is the most frequent malignant bone tumor in children and adolescents. Metastases are the major cause of death, and patients with relapse have poor prognosis. Several solid tumors display enhanced expression of matrix metalloproteinases (MMPs), and recently MMP-inhibitors have entered clinical trials. A disturbance of the MMP system in favor of enhanced proteolytic activity may be suspected in OS because OS growth is accompanied by both enhanced local bone destruction and bone formation, two processes that are dependant on proteolytic enzymes. Thus, the aim of the present study was to evaluate the involvement of MMPs in a panel of human OS cell lines, xenografts and biopsies.

Material and methods

Expression of MMPs and their endogenous inhibitors were studied by zymography and Northern blot analyses. In vitro invasion of OS cell lines and effects of MMP-inhibitors (Marimastat and doxycycline) were assessed in the transwell chamber assay.

Results

In vitro invasiveness was compared with gelatinase activity, and the most invasive cell line secreted the highest amounts of MMP-2 and MMP-9. Two different MMP-inhibitors significantly reduced OS cell invasion. The majority of the OS xenografts expressed both the inactive and active form of MMP-2 and in some cases also MMP-9. The biopsies from primary and metastatic OS also expressed MMP-2 mRNA. However, MMP-9 levels were higher in the biopsies than in the xenografts.

Conclusion

The obtained results support the hypothesis that MMPs and their endogenous inhibitors participate in the invasive process of human OS.

Introduction

In normal bone, matrix is constantly degraded and replaced by new matrix, a process requiring a proper balance between bone degradation and synthesis [1]. Proteolytic enzymes like matrix metalloproteinases (MMP), plasminogen activators and cathepsins are required in this process 2, 3, 4. Recent reports on MMP knockout mice and on human genetic disorders have drawn the attention to the importance of MMPs for normal skeletal development 5, 6. The human MMP family consists of more than 20 proteinases and can degrade various components of the bone matrix, like collagen, proteoglycans, fibronectin, and laminin. Furthermore, MMPs can induce alterations in the extracellular environment and thereby affect cell activities, for instance by release of TGF-β from the bone matrix and degradation of IL-β and calcitonin. The control systems regulating the biological activity of these proteinases are complex and occur at three different levels; transcription, activation of the enzymes from their latent to the active form and inhibition by endogenous inhibitors, like tissue inhibitors of MMPs (TIMPs) and plasminogen activator inhibitors.

Tumor growth, invasion, and metastasis are processes that include tumor cell proliferation, proteolytic digestion of the extracellular matrix (ECM), cell migration through basement membranes into the circulatory system, extravasation, and growth at the metastatic site [7]. MMPs are involved in the metastatic process as a consequence of their capacity to degrade basement membranes, thus facilitating invasion and metastasis. In addition, the MMPs can, by their proteolytic activity, promote tumor growth by increasing the bioavailability of growth factors residing in the ECM [8]. Furthermore, angiogenesis is a prerequisite for tumor growth, as all tumors larger than a few mm require their own blood supply, and tissue-remodeling enzymes are necessary for penetration of the newly formed blood vessels into the tumor [9]. Overexpression of MMPs in malignant tissue compared to corresponding normal tissue has been demonstrated in a large variety of malignant tumors like lung, colon, breast, prostate, and pancreatic carcinoma, and clinical evidence indicates that overproduction of MMPs confers a poor prognosis in several of these tumor types. In general, the gelatinases (MMP-2 and -9) are the two MMPs most consistently overexpressed in malignant tissues and, thus, associated with tumor aggressiveness, metastatic potential, and poor prognosis.

Osteosarcoma (OS) is a highly malignant bone tumor characterized by formation of neoplastic bone tissue and is the most frequent malignant bone tumor in children and adolescents 10, 11. Radiological evidence of both bone destruction and bone formation is characteristic, the latter representing neoplastic bone. Patients with localized disease are reported to have an event-free 5-year survival between 44% and 78%. Metastases, most commonly to the lungs, are the major cause of death, and patients with relapse are reported to have 5-year-survival rates between 14% and 50%. Additionally, a disturbing number of long-term survivors of OS have severe late effects, including second cancers and anthracycline-induced cardiomyopathy. Therefore, novel treatment modalities to ensure better outcome for OS patients are warranted.

A disturbance of the MMP/TIMP balance in favor of enhanced proteolytic activity may be suspected in OS since OS growth is accompanied by both enhanced local bone destruction and bone formation, two processes that are dependant on proteolytic enzymes. Thus, the aim of the present study was to evaluate the importance of MMPs and TIMPs in a panel of human OS cell lines, xenografts and biopsies. Given enhanced MMP activity in OS, inhibition of MMPs could be a target for treatment in OS patients.

Section snippets

In Vitro Cell Culture

The human OHS and KPDX cell lines were established at Department of Tumor Biology, The Norwegian Radium Hospital 12, 13, whereas the U2OS and SaOS cell lines were purchased from the American Type Culture Collection. The cells were cultivated in RPMI 1640 (Life technologies Inc., Middlesex, United Kingdom), supplemented with 10% heat-inactivated fetal calf serum (Life technologies Inc.) and l-glutamine (2.0 mm; Life technologies Inc.).

Animals and Establishment of Tumor Xenografts

Tumor tissue from altogether 21 patients treated for OS at

Gelatinolytic Activity and in Vitro Invasive Properties of Human OS Cell Lines

Zymography showed that all of the examined OS cell lines secreted MMP-2 protein into the culture media, while the active 62-kDa form of MMP-2 was only present at detectable levels in conditioned media from the OHS cells (Fig. 1). Activation of MMP-2 is thought to occur through an intermediate form [19], and the gelatinase of approximately 64–67 kDa secreted by the OHS cells probably represents the intermediate form of MMP-2. In contrast to the universal production of MMP-2, only the U2OS cell

Discussion

In the present study the expression of several MMPs and TIMPs were examined in a panel of human OS cell lines, xenografts and biopsies in an attempt to investigate the involvement of matrix degrading enzymes in this type of cancer. The in vitro invasiveness of the cell lines were compared with the activity of the gelatinases, and our analysis revealed that the most invasive cell line secreted the highest amounts of MMP-2 and MMP-9. The fact that two different MMP-inhibitors significantly

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    This study was financially supported by Stiftelsen Sophies Minde, The Norwegian Research Council, and The Norwegian Cancer Society.

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