Zhong Dong

Matrix Metalloproteinase Activity and Osteoclasts in Experimental Prostate Cancer Bone Metastasis Tissue

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 tumor-induced preosteoclast motility was dependent on MMP-9 expression. These data suggest that osteoclast-derived MMP-9 may represent a potential therapeutic target in bone metastasis and provide a rationale for the development of MMP-9-specific inhibitors.

CXCL12/CXCR4 Transactivates HER2 in Lipid Rafts of Prostate Cancer Cells and Promotes Growth of Metastatic Deposits in Bone

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. (Mol Cancer Res 2008;6(3):446–57)

Inhibition of human prostate cancer growth, osteolysis and angiogenesis in a bone metastasis model by a novel mechanism-based selective gelatinase inhibitor

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.

Differential regulation of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 expression in co-cultures of prostate cancer and stromal cells

Tumor–stromal interactions have been suggested to be a critical factor in both tumor invasion and tumor metastasis. Here, we examined the role of tumor–stromal interactions using co‐cultures of prostate cancer (PC) cells derived from primary and metastatic tumors with primary or immortalized stromal (fibroblast and smooth muscle) cells and their effect on matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) expression. Co‐cultures of PC and stromal cells showed enhanced levels of pro‐MMP‐9 and reduced levels of TIMP‐1 and TIMP‐2. Whereas enhanced expression of pro‐MMP‐9 occurred in PC cells, the TIMPs were down‐regulated in stromal cells. Induction of pro‐MMP‐9 and reduction of TIMP expression did not require cell–cell contact and were mediated by a soluble factor(s) present in the conditioned medium of the effector cell. Collagen I is a potent inducer of pro‐MMP‐9 in PC cells. Consistently, preliminary characterization of the soluble factor in the fibroblast conditioned medium revealed m.w. of approximately 100 to 250 kDa, and its effect on pro‐MMP‐9 expression was partly inhibited by an anti‐α2 integrin antibody, a major collagen I receptor. Expression of pro‐MMP‐9 protein and mRNA was also induced in metastatic PC‐3 cells grown in human fetal bone implants in SCID mice. Together, these findings demonstrate the importance of tumor–stromal interactions in the regulation of MMP and TIMP expression and their potential role in PC progression.

Bone marrow stromal cells enhance prostate cancer cell invasion through type I collagen in an MMP‐12 dependent manner

At the cellular level, the process of bone metastasis involves many steps. Circulating cancer cells enter the marrow, proliferate, induce neovascularization, and ultimately expand into a clinically detectable, often symptomatic, metastatic deposit. Although the initial establishment and later expansion of the metastatic deposit in bone require tumor cells to possess invasive capability, the exact proteases responsible for this phenotype are not well known. The objective of our study was to take an unbiased approach to determine which proteases were expressed and functional during the initial interactions between prostate cancer cells and bone marrow stromal (BMS) cells. We found that the combination of human prostate cancer PC3 and BMS cells stimulates the invasive ability of cancer cells through type I collagen. The use of inhibitors for each of the major protease families indicated that 1 or more MMPs was/were responsible for the BMS‐induced invasion. Gene profiling and semiquantitative RT‐PCR analysis revealed an increased expression of several MMP genes because of PC3/BMS cell interaction. However, only MMP‐12 showed an increase in protein expression. Downregulation of MMP‐12 expression in PC3 cells by siRNA inhibited the enhanced invasion induced by PC3/BMS cell interaction. In vivo, MMP‐12 was found to be primarily expressed by prostate cancer cells growing in bone. Our data suggest that BMS cells induce MMP‐12 expression in prostate cancer cells, which results in invasive cells capable of degradation of type I collagen.

Host matrix metalloproteinase-9 contributes to tumor vascularization without affecting tumor growth in a model of prostate cancer bone metastasis

Matrix metalloproteinases (MMPs) have been associated with initiation, progression and vascularization of a number of tumors. However, clinical trials using MMP inhibitors failed to meet expectations. Previously, we demonstrated the potential importance of MMP-9 activity in experimental prostate cancer bone tumor tissue. However, the particular roles of host- and tumor-derived MMP-9 remains to be defined. Herein, we examined the role of host MMP-9 in subcutaneous and intraosseous growth of the human androgen independent prostate cancer cell line PC3 in MMP-9 deficient mice. In the subcutaneous model, the tumor incidence in the control (RAG-1ko/ko) and experimental (RAG-1ko/ko /MMP-9ko/ko) group was 100%, with similar tumor growth kinetics and microvascular densities. In the intraosseous tumor model, the tumor incidence was higher in RAG-1ko/ko /MMP-9ko/ko mice than in RAG-1ko/ko mice (67% and 39%, respectively), though no statistical differences were found. The intraosseous tumor areas were similar in both groups, and the number of tumor-associated osteoclasts did not differ significantly. However, the microvascular density of intraosseous tumors was higher in RAG-1ko/ko than in RAG-1ko/ko/MMP-9ko/ko mice, though no changes in tumor growth could be detected. In an in vitro assay, we found that bone marrow (BM) cells increased the invasiveness of PC3 cells, and that this enhancement was independent of MMP-9 expression by marrow cells. Our results with the RAG-1 model suggest that host-derived MMP-9 is neither necessary nor sufficient for subcutaneous or intraosseous PC3 tumor growth, osteoclastic response, or in vitro invasiveness of tumor cells.

Immunohistochemical analysis of ocular platelet basic protein expression during infection with Pseudomonas aeruginosa.

Platelet basic protein (PBP) and several of its derivatives are known to express a wide range of biological characteristics. It is the precursor of connective tissue activating peptide (CTAP-III), beta thromboglobulin (beta-TG) and neutrophil activating peptide (NAP-2), which is the proteolytic derived end product. The temporal ocular expression of the chemokine PBP before and during corneal infection over several days by Pseudomonas aeruginosa was examined by immunohistochemistry. Prior to corneal infection, immunohistochemical staining demonstrated the constitutive expression of PBP in the cornea, lens and retina. PBP expression was present in the corneal epithelium, stromal fibroblasts and endothelium. There was a temporal increase in PBP expression in the cornea after infection. The entire cornea exhibited extensive cellular infiltration by positive PBP staining infiltrating cells within 6 days post-infection. The cornea, lens and retina underwent extensive degradation within 5-6 days post-infection with some apparent selective increase in PBP staining in the lens and retina.