Bone marrow-derived cathepsin K cleaves SPARC in bone metastasis.

Bone metastasis is a hallmark of advanced prostate and breast cancers, yet the critical factors behind attraction of tumors to the skeleton have not been validated. Here, we investigated the involvement of cathepsin K in the progression of prostate tumors in the bone, which occurs both by direct degradation of bone matrix collagen I and by cleavage of other factors in the bone microenvironment. Our results demonstrated that bone marrow-derived cathepsin K is capable of processing and thereby modulating SPARC, a protein implicated in bone metastasis and inflammation. The coincident up-regulation of SPARC and cathepsin K occurred both in vivo in experimental prostate bone tumors, and in vitro in co-cultures of bone marrow stromal cells with PC3 prostate carcinoma cells. PC3-bone marrow stromal cell interaction increased secretion and processing of SPARC, as did co-cultures of bone marrow stromal cells with two other cancer cell lines. In addition, bone marrow stromal cells that were either deficient in cathepsin K or treated with cathepsin K inhibitors had significantly reduced secretion and cleavage of SPARC. Increases in secretion of pro-inflammatory cytokines (ie, interleukin-6, -8) coincident with overexpression of cathepsin K suggest possible mechanisms by which this enzyme contributes to tumor progression in the bone. This is the first study implicating bone marrow cathepsin K in regulation of biological activity of SPARC in bone metastasis.

Hu/Mu ProtIn oligonucleotide microarray: dual-species array for profiling protease and protease inhibitor gene expression in tumors and their microenvironment.

Proteolysis is a critical regulatory mechanism for a wide variety of physiologic and pathologic processes. To assist in the identification of proteases, their endogenous inhibitors, and proteins that interact with proteases or proteolytic pathways in biological tissues, a dual-species oligonucleotide microarray has been developed in conjunction with Affymetrix. The Hu/Mu ProtIn microarray contains 516 and 456 probe sets that survey human and mouse genes of interest (proteases, protease inhibitors, or interactors), respectively. To investigate the performance of the array, gene expression profiles were analyzed in pure mouse and human samples (reference RNA; normal and tumor cell lines/tissues) and orthotopically implanted xenografts of human A549 lung and MDA-MB-231 breast carcinomas. Relative gene expression and "present-call" P values were determined for each probe set using dChip and MAS5 software, respectively. Despite the high level of sequence identity of mouse and human protease/inhibitor orthologues and the theoretical potential for cross-hybridization of some of the probes, >95% of the "present calls" (P<0.01) resulted from same-species hybridizations (e.g., human transcripts to human probe sets). To further assess the performance of the microarray, differential gene expression and false discovery rate analyses were carried out on human or mouse sample groups, and data processing methods to optimize performance of the mouse and human probe sets were identified. The Hu/Mu ProtIn microarray is a valuable discovery tool for the identification of components of human and murine proteolytic pathways in health and disease and has particular utility in the determination of cellular origins of proteases and protease inhibitors in xenograft models of human cancer.

Bone microenvironment modulates expression and activity of cathepsin B in prostate cancer.

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.

Cathepsin B and tumor proteolysis: contribution of the tumor microenvironment.

Tumor-stromal interactions induce expression of matrix metalloproteinases and serine proteases and, as shown recently, the cysteine protease cathepsin B. We speculate that such interactions upregulate the transcription factor Ets1, resulting in increased cathepsin B expression. This would be consistent with the observed concomitant upregulation of matrix metalloproteinases and serine proteases as well as with the ability of extracellular matrices and their binding partners to alter cathepsin B expression and secretion. Using a confocal assay to analyze the contribution of tumor-stromal interactions to proteolysis, we have been able to confirm enhanced degradation of extracellular matrices by all three classes of proteases.

Mutant K-ras regulates cathepsin B localization on the surface of human colorectal carcinoma cells.

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.