These data suggest that chemoattractive mechanisms involve migration of cancer cells towards bone tissue, and that cell signaling induced by binding of the chemokine to its receptor leads to the activation of multiple signaling pathways and subsequent secretion of MMP-9 into the local environment. These findings provide a link between chemoattractive mechanisms, growth of tumor cells in bone, and tumor-enhanced bone matrix turnover.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membranetype 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (K i ) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k on of 10 2 to 10 4 M ؊1 s ؊1 ), and the reversal of the process is slow (k off of 10 ؊3 to 10 ؊4 s ؊1 ). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.Extracellular proteolysis is an essential aspect of both physiological and pathological processes. Several enzyme families have been implicated in extracellular proteolysis, of which the matrix metalloproteinases (MMPs) 3 constitute an important group. The MMPs are zinc-dependent endopeptidases that play key roles in embryonic development, neurological processes, wound healing, angiogenesis, arthritis, cardiovascular diseases, and cancer, just to mention a few examples. In cancer, for instance, MMPs are implicated at all stages of tumor progression, including tumor growth, angiogenesis, and metastasis (1). Two MMPs, gelatinases A and B (MMP-2 and MMP-9, respectively), are highly expressed in human cancer, and a direct relationship between cancer progression and gelatinase expression and activity has been well established in many studies (2). As tumors manifest high levels of gelatinase activity, inhibitors specific for the gelatinases are highly sought.In the past 8 years, there have been numerous approaches aimed at targeting MMP activities in tumors, and several clinical trials were carried out to test the efficacy of various inhibitors. Unfortunately, the results of these trials were disappointing due to the lack of an objective clinical response and undesired side effects. Many reasons have been postulated for these effects, but at the core of the problem remains the issue of inhibitor ...

Previously, we and others showed that broad spectrum pharmaceutical inhibition of matrix metalloproteinase (MMP) activity reduces intraosseous tumor burden and bone degradation in animal models of bone metastasis. Herein, we used specific assays to measure net enzymatic activities of individual MMPs during colonization of bone by prostate cancer cells. PC3 cells were injected into the marrow of human fetal femurs previously implanted in SCID mice. Net MMP-9 activity in bone tissues peaked 2 weeks after injection, coinciding with a wave of osteoclast recruitment. In contrast, MMP-2 and MT1-MMP activity did not change. In vitro, co-culture of PC3 cells with bone tissue led to activation of pro-MMP-9 and increases in secreted net MMP-9 activity. Activation of pro-MMP-9 was prevented by metalloprotease inhibitors but not by inhibitors of other classes of proteases. Ribozyme suppression of MMP-9 expression in PC3 cells did not affect pro-MMP-9 activation or net MMP-9 activity and did not affect the phenotype of bone tumors. siRNA targeting of MMP-9 expression in preosteoclasts in vitro demonstrated that tumorinduced preosteoclast motility was dependent on MMP-9 expression. These data suggest that osteoclastderived MMP-9 may represent a potential therapeutic target in bone metastasis and provide a rationale for the development of MMP-9-specific inhibitors. Prostate cancer is the most common cancer and second leading cause of cancer death in American males.1 The overwhelming majority of prostate cancer deaths occurs in patients with metastases, and up to 90% of prostate cancer metastases occurs at skeletal sites. 2 Patients with bone metastasis frequently suffer from pain, pathological fractures, spinal cord compression, hypercalcemia, and bone marrow suppression.3,4 Although prostate cancer metastases typically appear osteosclerotic on radiographical imaging studies, multiple lines of evidence demonstrate clearly that both bone degradation and bone formation are present within the metastatic deposits. Importantly, there is now evidence that therapies that specifically target skeletal metastases, as opposed to general metastases, may extend survival in patients with prostate cancer.

Chemokines and their receptors function in migration and homing of cells to target tissues. Recent evidence suggests that cancer cells use a chemokine receptor axis for metastasis formation at secondary sites. Previously, we showed that binding of the chemokine CXCL12 to its receptor CXCR4 mediated signaling events resulting in matrix metalloproteinase-9 expression in prostate cancer bone metastasis. A variety of methods, including lipid raft isolation, stable overexpression of CXCR4, cellular adhesion, invasion assays, and the severe combined immunodeficient -human bone tumor growth model were used. We found that (a) CXCR4 and HER2 coexist in lipid rafts of prostate cancer cells; (b) the CXCL12/CXCR4 axis results in transactivation of the HER2 receptor in lipid rafts of prostate cancer cells; (c) Src kinase mediates CXCL12/CXCR4 transactivation of HER2 in prostate cancer cells; (d) a pan-HER inhibitor desensitizes CXCR4-induced transactivation and subsequent matrix metalloproteinase-9 secretion and invasion; (e) lipid raft -disrupting agents inhibited raft-associated CXCL12/CXCR4 transactivation of the HER2 and cellular invasion; (f) overexpression of CXCR4 in prostate cancer cells leads to increased HER2 phosphorylation and migratory properties of prostate cancer cells; and (g) CXCR4 overexpression enhances bone tumor growth and osteolysis. These data suggest that lipid rafts on the cell membrane are the key site for CXCL12/CXCR4 -induced HER2 receptor transactivation. This transactivation contributes to enhanced invasive signals and metastatic growth in the bone microenvironment.

ABSTRACT

Metastasis to the bone is a major clinical complication in patients with prostate cancer (PC). However, therapeutic options for treatment of PC bone metastasis are limited. Gelatinases are members of the matrix metalloproteinase (MMP) family and have been shown to play a key role in PC metastasis. Herein, we investigated the effect of SB-3CT, a covalent mechanism-based MMP inhibitor with high selectivity for gelatinases, in an experimental model of PC bone metastases. Intraperitoneal (i.p.) treatment with SB-3CT (50 mg/kg) inhibited intraosseous growth of human PC3 cells within the marrow of human fetal femur fragments previously implanted in SCID mice, as demonstrated by histomorphometry and Ki-67 immunohistochemistry. The anti-osteolytic effect of SB-3CT was confirmed by radiographic images. Treatment with SB-3CT also reduced intratumoral vascular density and bone degradation in the PC3 bone tumors. A direct inhibition of bone marrow endothelial cell invasion and tubule formation in Matrigel by SB-3CT in vitro was also demonstrated. The use of the highly selective gelatinase inhibitors holds the promise of effective intervention of metastases of PC to the bone. ' 2005 Wiley-Liss, Inc.Key words: matrix metalloproteinases; endothelium; MMP-2; MMP-9; gelatinase inhibitor PC is the second leading cause of cancer death in males in the United States. 1 Bone metastasis represents a major clinical complication of PC and is usually associated with pain, fractures and other life-impairing conditions. 2 In spite of the extent of this complication, the therapeutic options for the treatment of PC bone metastasis are very limited and are mostly palliative in nature. Therefore, new approaches to treat PC bone metastasis are urgently needed.Members of the matrix metalloproteinase (MMP) family of zincdependent endopeptidases, in particular gelatinases A and B, also known as MMP-2 and MMP-9, have been associated with the development of bone metastasis by PC cells. 3-8 Therefore, MMPs constitute an attractive target for intervention in PC bone metastasis. A limited number of preclinical studies reported the ability of synthetic MMP inhibitors to inhibit primary and metastatic PC growth in animal models. 9-11 We previously reported that administration of the broad-spectrum MMP inhibitor batimastat (BB-94) reduced tumor burden and bone degradation by human PC3 cells growing within human bone in severe combined immunodeficiency (SCID) mice. 8 These studies demonstrated the potential benefit of targeting MMP activity in PC bone metastasis. In spite of the success of broad-spectrum MMP inhibitors in preclinical studies, these inhibitors failed to demonstrate therapeutic efficacy in clinical trials in patients with advanced cancer, and also produced undesired side effects. [12][13][14] Lack of selectivity has been considered one of the main reasons for the disappointing performance of broad-spectrum MMP inhibitors in clinical trials. 12,14 Thus, targeting of specific and relevant MMPs in cancer progression remains an important goal.A...

The tyrosine kinase receptor c-kit and its ligand stem cell factor (SCF) have not been explored in prostate cancer (PC) bone metastasis. Herein, we found that three human PC cell lines and bone marrow stromal cells express a membrane-bound SCF isoform and release a soluble SCF. Bone marrow stromal cells revealed strong expression of c-kit, whereas PC cells showed very low levels of the receptor or did not express it all. Using an experimental model of PC bone metastasis, we found that intraosseous bone tumors formed by otherwise c-kit-negative PC3 cells strongly expressed c-kit, as demonstrated using immunohistochemical and Western blot analyses. Subcutaneous PC3 tumors were, however, c-kit-negative. Both bone and subcutaneous PC3 tumors were positive for SCF. Immunohistochemical analysis of human specimens revealed that the expression frequency of c-kit in epithelial cells was of 5% in benign prostatic hyperplasia, 14% in primary PC, and 40% in PC bone metastases, suggesting an overall trend of increased c-kit expression in clinical PC progression. Stem cell factor expression frequency was more than 80% in all the cases. Our data suggest that the bone microenvironment up-regulates c-kit expression on PC cells, favoring their intraosseous expansion.

Maspin is an epithelial-specific tumor suppressor gene. Previous data suggest that maspin expression may redirect poorly differentiated tumor cells to better differentiated phenotypes. Further, maspin is the first and only endogenous polypeptide inhibitor of histone deacetylase 1 (HDAC1) identified thus far. In the current study, to address what central program of tumor cell redifferentiation is regulated by maspin and how tumor microenvironments further define the effects of maspin, we conducted a systematic and extensive comparison of prostate tumor cells grown in 2-dimensional culture, in 3-dimensional collagen I culture, and as in vivo bone tumors. We showed that maspin was sufficient to drive prostate tumor cells through a spectrum of temporally and spatially polarized cellular processes of redifferentiation, a reversal of epithelial-to-mesenchymal transition (EMT). Genes commonly regulated by maspin were a small subset of HDAC target genes that are closely associated with epithelial differentiation and TGFβ signaling. These results suggest that a specific endogenous HDAC inhibitor may regulate one functionally related subset of HDAC target genes, although additional maspin-induced changes of gene expression may result from tumor interaction with its specific microenvironments. Currently, EMT is recognized as a critical step in tumor progression. To this end, our current study uncovered a link between maspin and a specific mechanism of prostate epithelial differentiation that can reverse EMT.