Identification of activated matrix metalloproteinase-2 (MMP-2) as the main gelatinolytic enzyme in malignant melanoma by in situ zymography

Abstract

The involvement of extracellular matrix-degrading enzymes, such as matrix metalloproteinases and serine proteases, during tumour progression and metastasis is well established. In particular, the activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP has been shown to contribute to the invasive abilities of various tumours. This study presents evidence that in tissue of malignant melanomas, increased effective gelatinolytic activity is mainly located at sites where melanoma cells interact with the surrounding extracellular matrix. Forty-one primary melanomas (30 superficial spreading and 11 nodular type) and six lymph node metastases were investigated by a modified technique of gelatin in situ zymography. This technique localizes areas of effective proteolytic activity within tissue sections. In 28/41 (68%) primary melanomas and in 6/6 (100%) metastases, considerable proteolysis was detected at the invading part of the tumour and especially at sites of tumour-stroma interactions, whereas no or only weak proteolytic activity was localized within the centres of solid nests of tumour cells. Zymographic analysis of extracts obtained from different areas of microdissected melanoma specimens identified activated MMP-2 as the enzyme responsible for this activity. Immunohistochemical analysis detected strong staining for MMP-2 and MT1-MMP, even in areas in which no proteolytic activity was found by in situ zymography, emphasizing the importance of more functional techniques for the investigation of balanced proteolytic systems. This technology makes it possible to draw conclusions regarding the balance between activated proteases and inhibitors, which are frequently found to be present together in close proximity in vivo.