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 alphavbeta3 correlate with
melanoma progression. Recently, direct binding of
matrix metalloproteinase-2 to alpha(v)beta3 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)beta3-negative
melanoma cell lines MV3 and BLM, their beta3-transfected alpha(v)beta3 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)beta3-negative and alpha(v)beta3-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)beta3-negative cell lines and undetectable in the alpha(v)beta3-positive cell lines. Conversely, active
matrix metalloproteinase-2 was present exclusively on the cell surface of the alpha(v)beta3 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 beta3 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)beta3 expressing MV3 and BLM cell lines. In human cutaneous
melanoma lesions, neither
matrix metalloproteinase-2 nor
integrin alpha(v)beta3 is detectable in
melanoma in situ as determined by immunohistochemistry. In contrast, the number of matrix metalloproteinase-2-positive and alphavbeta3-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)beta3 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 alphavbeta3-double-positive
melanoma cells were detectable. Our study demonstrates that the presence of activated
matrix metalloproteinase-2 correlates with expression of alpha(v)beta3 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.