R. Daniel Bonfil

Potent Mechanism-based Inhibitors for Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membrane-type 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 (Ki) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (kon of 102 to 104 M-1 s-1 and the reversal of the process is slow (koff 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.

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)

A Potential Oncogenic Activity of Platelet-Derived Growth Factor D in Prostate Cancer Progression

The platelet-derived growth factor (PDGF) proteins are potent stimulators of cell proliferation/transformation and play a major role in cell-cell communication. For over two decades, PDGFs were thought to exist as three dimeric polypeptides (the homodimers AA and BB and the heterodimer AB). Recently, however, the PDGF C and D chains were discovered in a BLAST search of the expressed sequence tag databases. The PDGF CC and DD dimers have a unique two-domain structure with an NH2-terminal CUB (compliment subcomponents C1r/C1s, Uegf, and Bmp1) domain and a COOH-terminal PDGF/vascular endothelial growth factor domain. Whereas secreted PDGF AA, BB, and AB readily activate their cell surface receptors, it was suggested that extracellular proteolytic removal of the CUB domain is required for the PDGF/vascular endothelial growth factor domain of PDGF CC and DD to activate PDGF receptors. In the present study, we examined the processing of latent PDGF D into its active form and the effects of PDGF D expression on prostate cancer progression. We show that LNCaP cells auto-activate latent PDGF DD into the active PDGF domain, which can induce phosphorylation of the β-PDGF receptor and stimulates LNCaP cell proliferation in an autocrine manner. Additionally, LNCaP-PDGF D-conditioned medium induces migration of the prostate fibroblast cell line 1532-FTX, indicating LNCaP-processed PDGF DD is active in a paracrine manner as well. In a severe combined immunodeficient mouse model, PDGF DD expression accelerates early onset of prostate tumor growth and drastically enhances prostate carcinoma cell interaction with surrounding stromal cells. These demonstrate a potential oncogenic activity of PDGF DD in the development and/or progression of prostate cancer.

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.

Shedding of RANKL by Tumor-Associated MT1-MMP Activates Src-Dependent Prostate Cancer Cell Migration

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays an essential role in protease-mediated extracellular matrix (ECM) degradation, but it also functions as a sheddase releasing non-ECM substrates such as receptor activator of NF-kappaB ligand (RANKL), an osteoclastogenic factor typically confined to the surface of osteoblasts. We previously found high expression of MT1-MMP in skeletal metastasis of prostate cancer patients, in a pattern similar to RANKL expression. We also showed that overexpression of MT1-MMP in prostate cancer cells increases tumor growth and osteolysis in an intratibial mouse model of bone metastasis, and that soluble factor(s) shed by tumor-derived MT1-MMP enhance osteoclast differentiation in a RANKL-dependent manner. Recent evidence indicates that the cognate receptor for RANKL, RANK, is expressed in prostate cancer cells, suggesting the presence of an autocrine pathway. In this study, we show that MT1-MMP-expressing LNCaP prostate cancer cells display enhanced migration. Moreover, conditioned medium from LNCaP cells expressing both RANKL and MT1-MMP stimulates the migration of MT1-MMP-deficient C42b prostate cancer cells. This enhanced chemotaxis can be abrogated by osteoprotegerin (soluble decoy receptor of RANKL), MIK-G2 (a selective inhibitor for MT1-MMP), and PP2 (a Src inhibitor). These findings indicate that tumor-derived MT1-MMP enhances tumor cell migration through initiation of an autocrine loop requiring ectodomain shedding of membrane-bound RANKL in prostate cancer cells, and that Src is a key downstream mediator of RANKL-induced migration of prostate cancer cells.

Higher antitumor activity of vinflunine than vinorelbine against an orthotopic murine model of transitional cell carcinoma of the bladder

The aim of this report was to investigate the feasibility of systemic treatment of transitional cell carcinoma of the bladder with vinflunine (VFL), and to compare its activity in respect to vinorelbine (VRL). Exposure of MB49 murine bladder cancer cells to both drugs showed a higher chemosensitivity of the cells to VRL than to VFL (IC50 values of 60 nM and 400 nM, respectively). Pretreatment of MB49 cells with non-cytotoxic drug concentrations revealed an inhibition of control in vitro invasiveness of 40 to 70% (1-25 nM VRL) and 22 to 80% (1-100 nM VFL) (P < 0.0001, ANOVA). The intraperitoneal administration of the drugs twice a week for 4 weeks in C57B1/6 female mice revealed that VFL was very well tolerated, with a 8-fold increase in the maximum tolerated dose in respect to VRL (40 mg/kg and 4.8 mg/kg, respectively). The administration schedule was evaluated in C57B1/6 female mice inoculated transurethraly with 5 x 10(4) MB49 cells. Intravesical tumor incidence on day 21 was 0% and 17% in mice treated intraperitoneally with 20 and 10 mg/kg VFL respectively (P = 0.0017 and P = 0.0001, Fischers Exact Test), contrasting with 75-83% obtained in all VRL-treated groups and Controls. All mice treated with 20 mg/kg VFL were still alive 60 days after intravesical MB49 tumor implantation, as well as 50% of those treated with 10 mg/kg VFL, while most of the remaining mice (Control and VRL-treated) died before day 32. These studies clearly demonstrate the activity of VFL against a murine bladder cancer model, with a favorable toxicity profile.

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.

Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection

This protocol requires 2–4 h and presents a method for injecting tumor cells, cancer stem cells or dispersed biopsy material into subcutaneous or orthotopic locations within recipient mice. The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4 °C and then injected into recipient animals at preferred anatomical sites. Tumor cells can also be co-injected with additional cell types, such as fibroblasts, stromal cells, endothelial cells and so on. Details are given on appropriate cell numbers, handling and concentration of the basement membrane proteins, recipient animals, injection location and techniques. This procedure enables the growth of tumors from cells or biopsy material (tumor graft) with greater efficiency of take and growth, and with retention of the primary tumor phenotype based on histology. Co-injection with additional cell types provides more physiological models of human cancers for use in drug screening and studying cancer biology.

Studies on the Mechanisms Responsible for Inhibition of Experimental Metastasis of B16-F10 Murine Melanoma by Pentoxifylline

Pentoxifylline (PTX), a methylxanthine derivative widely used as a hemorheological agent in the treatment of peripheral vascular disease, was studied to unveil the mechanisms responsible for its inhibitory action on B16-F10 experimental metastasis. In vitro pretreatment of B16-F10 cells with noncytotoxic concentrations of PTX significantly inhibited their adhesion to reconstituted basement membrane Matrigel(R) and type IV collagen as well as the relative activity of secreted 92 kD metalloproteinase. However, PTX pretreatment of B16-F10 cells did not affect their in vitro invasiveness. Heterotypic organ adhesion assays carried out with B16-F10 cells and suspended organ tissues demonstrated that pretreatment with noncytotoxic concentrations of PTX of both, tumor cells or lung tissue, brought about a dose-dependent inhibition of melanoma cell adhesion to lung. Immunohistochemical studies using antibodies against CD31 adhesion molecule (PECAM-1) revealed that B16-F10 cells adhere to lung endothelial cells. Our results suggest that PTX may exert its inhibitory effect on tumor lodgment, and as a consequence of that on experimental metastases, through an inhibitory action on cell adhesion molecules.