Lysosomal cathepsin B has been implicated in parasitic, inflammatory and neoplastic diseases. Most of these pathologies suggest a role for cathepsin B outside the cells, although the origin of extracellular active enzyme is not well defined. The activity of extracellular cathepsin B is difficult to assess because of the presence of inhibitors and inactivation of the enzyme by oxidizing agents. Therefore, we have developed a continuous assay for measurement of cathepsin B activity produced pericellularly by living cells. The kinetic rate of Z-Arg-Arg-NHMec conversion was monitored and the assay optimized for enzyme stability, cell viability and sensitivity. To validate the assay, we determined that human liver cathepsin B was stable and active under the conditions of the assay and its activity could be inhibited by the selective epoxide derivative CA-074. Via this assay, we were able to demonstrate that active cathepsin B was secreted pericellularly by viable cells. Both preneoplastic and malignant cells secreted active cathepsin B. Pretreatment of cells with the membranepermeant proinhibitor CA-074Me completely abolished pericellular and total cathepsin B activity whereas pretreatment with the active drug CA-074 had no effect. Immunoprecipitation and immunoblotting experiments suggested that the active enzyme species was 31-kDa single-chain cathepsin B. Exocytosis of cathepsin B was not related to secretion of proenzyme or secretion from mature lysosomes. Our results suggest an alternative pathway for exocytosis of active cathepsin B.

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Prostate and breast cancers commonly metastasize to skeletal sites and locally disrupt normal bone remodeling.Despite recent progress in cancer detection and treatment, it remains unclear which skeletal-specific factors are among the critical determinants in preferential localization of metastatic cells to the bone. Recent clinical and experimental data suggest that accelerated bone remodeling may be responsible for homing of tumor cells to the bone.1,2 This is evidenced by the increased metastasis in response to experimental treatment with calciotropic hormone or to androgen ablation 1,2 and reduced incidence of metastasis with antiresorptive therapies.3 Establishment of tumors in bone requires multidirectional interactions between tumor cells, bone cells, stromal cells, and inflammatory components, as well as extracellular matrix proteins. This complex interplay between tumor cells and the bone microenvironment facilitates increased bone turnover and promotes tumor cell survival.The key enzyme responsible for osteolysis of bone is the cysteine protease cathepsin K, which is the only known mammalian protease capable of degrading both the helical and non-helical regions of collagen I, the main component of the organic bone matrix. 4 Within the bone microenvironment, cathepsin K localizes predominantly to osteoclasts and its overexpression results in increased bone turnover.5 Accordingly, a deficiency in this potent collagenase results in a bone-sclerosing disorder called pycnodysostosis in man and osteopetrosis in mice. 6,7 The presence of cathepsin K has been demonstrated in many malignancies including prostate and breast cancers, both of which have a high propensity to metastasize to bone. 8 -10 A role for cathepsin K in advanced cancers has been attributed mainly to its ability to degrade native collagen I and facilitate the expansion of tumors in the bone. Our recent studies and data by other groups suggest that cathepsin K also cleaves and thereby modulates the biological activity of several important proteins in the bone microenvironment.11-15 Of particular importance is Supported by grants from the DOD PC030325. DOD PC074031. DOD

Cathepsin B protein and activity are known to localize to the basal plasma membrane of colon carcinoma cells following the appearance of K-ras mutations. Using immunofluorescence and subcellular fractionation techniques and two human colon carcinoma cell lines - one with a mutated K-ras allele (HCT 116) and a daughter line in which the mutated allele has been disrupted (HKh-2)-we demonstrate that the localization of cathepsin B to caveolae on the surface of these carcinoma cells is regulated by mutant K-ras. In HCT 116 cells, a greater percentage of cathepsin B was distributed to the caveolae, and the secretion of cathepsin B and pericellular (membrane-associated and secreted) cathepsin B activity were greater than observed in HKh-2 cells. Previous studies established the light chain of annexin II tetramer, p11, as a binding site for cathepsin B on the surface of tumor cells. The deletion of active K-ras in HKh-2 cells reduced the steady-state levels of p11 and caveolin-1 and the distribution of p11 to caveolae. Based upon these results, we speculate that cathepsin B, a protease implicated in tumor progression, plays a functional role in initiating proteolytic cascades in caveolae as downstream components of this cascade (e.g., urokinase plasminogen activator and urokinase plasminogen activator receptor) are also present in HCT 116 caveolae.

Prostate cancers metastasize to bone leading to osteolysis. Here we assessed proteolysis of DQ-collagen I (a bone matrix protein) and, for comparison, DQ-collagen IV, by living human prostate carcinoma cells in vitro. Both collagens were degraded, and this degradation was reduced by inhibitors of matrix metallo, serine, and cysteine proteases. Because secretion of the cysteine protease cathepsin B is increased in human breast fibroblasts grown on collagen I gels, we analyzed cathepsin B levels and secretion in prostate cells grown on collagen I gels. Levels and secretion were increased only in DU145 cells--cells that expressed the highest baseline levels of cathepsin B. Secretion of cathepsin B was also elevated in DU145 cells grown in vitro on human bone fragments. We further investigated the effect of the bone microenvironment on cathepsin B expression and activity in vivo in a SCID-human model of prostate bone metastasis. High levels of cathepsin B protein and activity were found in DU145, PC3, and LNCaP bone tumors, although the PC3 and LNCaP cells had exhibited low cathepsin B expression in vitro. Our results suggest that tumor-stromal interactions in the context of the bone microenvironment can modulate the expression of the cysteine protease cathepsin B.