Coexpression of integrin αvβ3 and matrix metalloproteinase-2 (MMP-2) coincides with MMP-2 activation:: Correlation with melanoma progression

Tumor cell invasion and metastasis formation depend on both adhesive and proteolytic mechanisms. Previous studies have shown that expression of matrix metalloproteinase-2 and integrin alpha(v)beta(3) correlate with melanoma progression. Recently, direct binding of matrix metalloproteinase-2 to alpha(v)beta(3) was implicated in presenting activated matrix metalloproteinase-2 on the cell surface of invasive cells. In this study we investigated this, using the highly metastatic, alpha(v)beta(3)-negative melanoma cell. lines MV3 and BLM, their beta(3)-transfected alpha(v)beta(3) expressing counterparts, xenografts derived from these cell lines, and fresh human cutaneous melanoma lesions comprising all stages of melanoma progression. Expression and activation status of matrix metalloproteinase-2 were studied by reverse transcription-polymerase chain reaction, immunohistochemistry, western blotting, and zymographic analysis, respectively. Matrix metalloproteinase-2 protein expression in vitro was similar in both alpha(v)beta(3)-negative and alpha(v)beta(3)-positive cell lines. Remarkable differences, however, exist in the localization of inactive and active matrix metalloproteinase-2. Soluble active matrix metalloproteinase-2 was detectable only in the conditioned medium of alpha(v)beta(3)-negative cell lines and undetectable in the alpha(v)beta(3)-positive cell lines. Conversely, active matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta(3) expressing transfectants, Western blot analysis of other components that are involved in matrix metalloproteinase-2 activation showed that processing of proMT1-matrix metalloproteinase to the activated form was enhanced in beta(3) transfectants, whereas secretion of tissue inhibitor of metalloproteinase-2 was decreased. In vivo, the presence of functionally active matrix metalloproteinase-2 was significantly higher in xenografts derived from the alpha(v)beta(3) expressing MV3 and BLM cell lines. In human cutaneous melanoma lesions, neither matrix metalloproteinase-2 nor integrin alpha(v)beta(3) is detectable in melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alpha(v)beta(3)-positive tumor cells was clearly increased in primary melanomas, and melanoma metastases. Double staining experiments and confocal laser microscopy demonstrated that the percentage of cells coexpressing matrix metalloproteinase-2 and alpha(v)beta(3) increased in advanced primary melanomas and melanoma metastases. In addition, zymography showed that functionally active matrix metalloproteinase-2 was frequently present in melanoma metastases. In these lesions a high proportion of matrix metalloproteinase-2- and alpha(v)beta(3)-double-positive melanoma cells were detectable. Our study demonstrates that the presence of activated matrix metalloproteinase-2 correlates with expression of alpha(v)beta(3) in human melanoma cells both in vitro and in vivo, and also in fresh human melanoma lesions. These findings strongly suggest that co-ordinated expression of both factors may be required for melanoma cell invasion and metastasis formation.