A LIPOXYGENASE METABOLITE, 12-(S)-HETE, STIMULATES PROTEIN-KINASE C-MEDIATED RELEASE OF CATHEPSIN-B FROM MALIGNANT-CELLS

The process of tumor cell invasion of the basement membrane is propose d to consist of three steps: attachment, local proteolysis and migrati on. 12-(S)-HETE, a 12-lipoxygenase metabolite of arachidonic acid, upr egulates surface expression of integrin cytoadhesins and an autocrine motility factor receptor, suggesting that this metabolite may play an important regulatory function in tumor cell invasion. In the present s tudy, we determined whether 12-(S)-HETE affects surface expression and /or release of cathepsin B, a cysteine protease that has been implicat ed in focal degradation of basement membrane. Secretion and distributi on of cathepsin B was evaluated in two model systems for various stage s of neoplastic progression: (i) murine B16 melanoma lines of low (B16 -F1) and high (B16a) lung colonization potential, and (ii) immortalize d and ras-transfected MCF-10 human breast epithelial cells that differ in their invasive capacities in vitro. In the B16a cells, 12-(S)-HETE induced release of native and latent cathepsin B activity and concomi tantly reduced cell-associated cathepsin B immunoreactivity. In contra st, 12-(S)-HETE did not induce the release of cathepsin B from B16-F1 cells, suggesting that there may be an enhanced response to 12-(S)-HET E in more malignant cells. This was confirmed in the MCF-10 system, in which 12-(S)-HETE was able to induce the release of cathepsin B from the ras-transfected cells, but not from the immortal cells. A simultan eous reduction in staining for cathepsin B was observed in the ras-tra nsfected cells, but not in their immortal counterparts. The release of cathepsin B may be mediated by PKC as pretreatment of B16a cells with the selective PKC inhibitor calphostin C, but not with the PKA inhibi tor HS, prevented the stimulated release of cathepsin B. In B16a cells , the release of cathepsin B was accompanied by a translocation toward the cell periphery of vesicles staining for cathepsin B, resulting in focal areas of accumulation of cathepsin B. After 12-(S)-HETE stimula tion of the ras-transfected MCF-10 cells, cathepsin B was distributed homogeneously on the apical surface. Thus, 12-(S)-HETE can upregulate the surface expression on tumor cells of proteins able to mediate each of the three steps of tumor cell invasion: adhesion, degradation, and migration. (C) 1994 Academic Press, Inc.