Dysregulation of fibulin-5 and matrix metalloproteases in epithelial ovarian cancer.

Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility. Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC). FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary. Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression. FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC. We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5. MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC. Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors. This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment. Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells. Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

Nephronectin Regulates Mesangial Cell Adhesion and Behavior in Glomeruli.

A critical aspect of kidney function occurs at the glomerulus, the capillary network that filters the blood. The glomerular basement membrane (GBM) is a key component of filtration, yet our understanding of GBM interactions with mesangial cells, specialized pericytes that provide structural stability to glomeruli, is limited. We investigated the role of nephronectin (Npnt), a GBM component and known ligand of α8ß1 integrin. Immunolocalization and in situ hybridization studies in kidneys of adult mice revealed that nephronectin is produced by podocytes and deposited into the GBM. Conditional deletion of Npnt from nephron progenitors caused a pronounced increase in mesangial cell number and mesangial sclerosis. Nephronectin colocalized with α8ß1 integrin to novel, specialized adhesion structures that occurred at sites of mesangial cell protrusion at the base of the capillary loops. Absence of nephronectin disrupted these adhesion structures, leading to mislocalization of α8ß1. Podocyte-specific deletion of Npnt also led to mesangial sclerosis in mice. These results demonstrate a novel role for nephronectin and α8ß1 integrin in a newly described adhesion complex and begin to uncover the molecular interactions between the GBM and mesangial cells, which govern mesangial cell behavior and may have a role in pathologic states.

Role of CD34 family members in lumen formation in the developing kidney.

Previous studies have shown CD34 family member Podocalyxin is required for epithelial lumen formation in vitro. We demonstrate that Endoglycan, a CD34 family member with homology to Podocalyxin, is produced prior to lumen formation in developing nephrons. Endoglycan localizes to Rab11-containing vesicles in nephron progenitors, and then relocalizes to the apical surface as progenitors epithelialize. Once an apical/luminal surface is formed, Endoglycan (and the actin-binding protein Ezrin) localize to large, intraluminal structures that may be vesicles/exosomes. We generated mice lacking Endoglycan and found mutants had timely initiation of lumen formation and continuous lumens, similar to controls. Mice with conditional deletion of both Endoglycan and Podocalyxin in developing nephrons also had normal tubular lumens. Despite this, Endoglycan/Podocalyxin is required for apical recruitment of the adaptor protein NHERF1, but not Ezrin, in podocyte precursors, a subset of the epithelia. In summary, while CD34 family members appear dispensable for lumen formation, our data identify Endoglycan as a novel pre-luminal marker and suggest lumen formation occurs via vesicular trafficking of apical cargo that includes Endoglycan.

ABT-263, a Bcl-2 inhibitor, enhances the susceptibility of lung adenocarcinoma cells treated with Src inhibitors to anoikis.

The tyrosine kinase Src plays an important role in the development of anoikis resistance in lung adenocarcinomas. Several suspension lung adenocarcinoma cell lines, which express phosphorylated Src, undergo apoptosis, or anoikis, in the presence of Src kinase inhibitors. However, lung adenocarcinoma cell lines vary in their sensitivity to Src inhibitors. We hypothesized that the addition of ABT-263, a potent Bcl-2 inhibitor, should significantly enhance the degree of anoikis in lung adenocarcinoma cells treated with Src inhibitors. In this study, we treated four suspension lung adenocarcinoma cell lines with ABT-263, an Src inhibitor (bosutinib or PP1), or a combination of both. In LC-KJ and HCC827 cells, combined treatment with ABT-263 and an Src inhibitor effectively induced anoikis, and the extent of anoikis was significantly greater than that induced by each agent alone; the synergy between the two drugs was apparent. Although we did not observe a marked increase in anoikis in LC-KJ and HCC827 cells treated with ABT-263 alone, H1650 and H1975 cells treated with ABT-263 (1 µM) upon detachment significantly underwent apoptosis, the levels of which were much greater than those observed upon attachment. However, the levels of anoikis induced by combination treatment were still greater than those by the individual agents in H1650 and H1975 cells. These findings provide a biological rationale to test combination therapy with ABT-263 and Src inhibitors in patients with lung adenocarcinoma.

Matrilysin (MMP-7) cleaves C-type lectin domain family 3 member A (CLEC3A) on tumor cell surface and modulates its cell adhesion activity.

Matrilysin (MMP-7) plays important roles in tumor progression. Previous studies have suggested that MMP-7 binds to tumor cell surface and promotes their metastatic potential. In this study, we identified C-type lectin domain family 3 member A (CLEC3A) as a membrane-bound substrate of MMP-7. Although this protein is known to be expressed specifically in cartilage, its message was found in normal breast and breast cancer tissues as well as breast and colon cancer cell lines. Because few studies have been done on CLEC3A, we overexpressed its recombinant protein in human cancer cells. CLEC3A was found in the cell membrane, extracellular matrix (ECM), and culture medium of the CLEC3A-expressing cells. CLEC3A has a basic sequence in the NH(2)-terminal domain and showed a strong heparin-binding activity. MMP-7 cleaved the 20-kDa CLEC3A protein, dividing it to a 15-kDa COOH-terminal fragment and an NH(2)-terminal fragment with the basic sequence. The 15-kDa fragment no longer had heparin-binding activity. Treatment of the CLEC3A-expressing cells with MMP-7 released the 15-kDa CLEC3A into the culture supernatant. Furthermore, the 20-kDa CLEC3A promoted cell adhesion to laminin-332 and fibronectin substrates, but this activity was abrogated by the cleavage by MMP-7. These results suggest that CLEC3A binds to heparan sulfate proteoglycans on cell surface, leading to the enhancement of cell adhesion to integrin ligands on ECM. It can be speculated that the cleavage of CLEC3A by MMP-7 weakens the stable adhesion of tumor cells to the matrix and promotes their migration in tumor microenvironments.

Matrilysin (matrix metalloprotease-7) cleaves membrane-bound annexin II and enhances binding of tissue-type plasminogen activator to cancer cell surfaces.

Matrilysin (matrix metalloproteinase-7) plays important roles in tumor progression. It was previously found that matrilysin binds to the surface of colon cancer cells to promote their metastatic potential. In this study, we identified annexin II as a novel membrane-bound substrate of matrilysin. Treatment of human colon cancer cell lines with active matrilysin released a 35 k Da annexin II form, which lacked its N-terminal region, into the culture supernatant. The release of the 35 k Da annexin II by matrilysin was significantly enhanced in the presence of serotonin or heparin. Matrilysin hydrolyzed annexin II at the Lys9-Leu10 bond, thus dividing the protein into an N-terminal nonapeptide and the C-terminal 35 k Da fragment. Annexin II is known to serve as a cell surface receptor for tissue-type plasminogen activator (tPA). Although the matrilysin treatment liberated the 35 k Da fragment of annexin II from the cell surface, it significantly increased tPA binding to the cell membrane. A synthetic N-terminal nonapeptide of annexin II bound to tPA more efficiently than intact annexin II. This peptide formed a heterodimer with intact annexin II in test tubes and on cancer cell surfaces. These and other results suggested that the nonapeptide generated by matrilysin treatment might be anchored to the cell membrane, possibly by binding to intact annexin II, and interact with tPA via its C-terminal lysine. It is supposed that the cleavage of cell surface annexin II by matrilysin contributes to tumor invasion and metastasis by enhancing tPA-mediated pericellular proteolysis by cancer cells.

Binding of active matrilysin to cell surface cholesterol sulfate is essential for its membrane-associated proteolytic action and induction of homotypic cell adhesion.

Regulation of cell surface molecules by matrix metalloproteinases (MMPs), as well as MMPs-catalyzed degradation of extracellular matrix, is important for tumor invasion and metastasis. Our previous study (Kioi, M., Yamamoto, K., Higashi, S., Koshikawa, N., Fujita, K., and Miyazaki, K. (2003) Oncogene 22, 8662-8670) demonstrated that active matrilysin specifically binds to the surface of colon cancer cells and induces notable cell aggregation due to processing of the cell membrane protein(s). Furthermore, these aggregated cells showed a dramatically enhanced metastatic potential. To elucidate the mechanism of matrilysin-induced cell aggregation, we attempted to identify the matrilysin-binding substance on the cell surface. Here, we demonstrate that cholesterol sulfate on the cell surface is a major matrilysin-binding substance. We found that active matrilysin bound to the cell membrane and cholesterol sulfate incorporated into liposomes with similar affinities. Treatment of colon cancer cells with beta-cyclodextrin significantly reduced not only matrilysin binding to the cell surface but also matrilysin-dependent proteolysis and cell aggregation. Interestingly, replenishment of cholesterol sulfate, but not cholesterol, neutralized the effects of beta-cyclodextrin. Taken together, it is likely that binding of matrilysin to cholesterol sulfate facilitates the matrilysin-catalyzed modulation of cell surface proteins, thus inducing the cancer cell aggregation.