Shouichi Higashi

Matriptase activates stromelysin (MMP-3) and promotes tumor growth and angiogenesis.

Matriptase/MT‐SP1, a type II membrane serine protease widely expressed in normal epithelial cells and human carcinoma cells, is thought to be involved in cancer progression. To clarify this possibility, we overexpressed exogenous matriptase in the human stomach cancer cell line AZ521. In vitro, the matriptase transfectant (Mat‐AZ521) and the control transfectant (Mock‐AZ521) showed a similar growth rate, although the saturation cell density was significantly higher with the Mat‐AZ521. When implanted into nude mice subcutaneously or intraperitoneally, Mat‐AZ521 cells grew faster and produced much larger solid tumors than Mock‐AZ521 cells. The overexpression of matriptase in AZ521 cells shortened the survival time of tumor‐bearing mice. Histological analysis showed that both the number and the size of blood vessels in tumor tissues were significantly higher in the Mat‐AZ521 tumors than the Mock‐AZ521 ones. Moreover, it was found that purified matriptase activated one of the important matrix metalloproteinases, stromelysin (MMP‐3). These results suggest the possibility that the matriptase‐dependent activation of MMP‐3, as well as the direct activity of matriptase, promotes tumor growth and angiogenesis by enhancing extracellular matrix degradation in tumor cell microenvironments. (Cancer Sci 2006; 97: 1327–1334)

Reactive Site-modified Tissue Inhibitor of Metalloproteinases-2 Inhibits the Cell-mediated Activation of Progelatinase A

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is supposed to play a regulatory role in the cell-mediated activation of progelatinase A. To investigate the mechanism of the regulation, we prepared and characterized a chemically modified TIMP-2, and examined its effects on the activation of progelatinase A. We found that treatment of TIMP-2 with cyanate ion led to loss of inhibitory activity toward matrilysin or gelatinase A. Structural and functional analyses of the modified TIMP-2 showed that carbamylation of the α-amino group of the NH2-terminal Cys1 of TIMP-2 led to complete loss of the inhibitory activity. When the reactive-site modified TIMP-2 was added to culture medium of concanavalin A-stimulated HT1080 cells, the conversion of endogenous progelatinase A to the intermediate form was partially inhibited, whereas that of the intermediate form to the mature one was strongly inhibited. The reactive site-modified TIMP-2 also prevented an accumulation of active gelatinase A on the cell surface. We speculate that occupation of the hemopexin-like domain of gelatinase A by the reactive site-modified TIMP-2 makes it unable for gelatinase A to be retained on the cell surface, thus preventing the autocatalytic conversion of the intermediate form of gelatinase A to its mature form.

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 β-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 β-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.

Identification of membrane-bound serine proteinase matriptase as processing enzyme of insulin-like growth factor binding protein-related protein-1 (IGFBP-rP1/angiomodulin/mac25)

Insulin‐like growth factor (IGF) binding protein‐related protein‐1 (IGFBP‐rP1) modulates cellular adhesion and growth in an IGF/insulin‐dependent or independent manner. It also shows tumor‐suppressive activity in vivo. We recently found that a single‐chain IGFB‐rP1 is proteolytically cleaved to a two‐chain form by a trypsin‐like, endogenous serine proteinase, changing its biological activities. In this study, we attempted to identify the IGFBP‐rP1‐processing enzyme. Of nine human cell lines tested, seven cell lines secreted IGFBP‐rP1 at high levels, and two of them, ovarian clear cell adenocarcinoma (OVISE) and gastric carcinoma (MKN‐45), highly produced the cleaved IGFBP‐rP1. Serine proteinase inhibitors effectively blocked the IGFBP‐rP1 cleavage in the OVISE cell culture. The conditioned medium of OVISE cells did not cleave purified IGFBP‐rP1, but their membrane fraction had an IGFBP‐rP1‐cleaving activity. The membrane fraction contained an 80‐kDa gelatinolytic enzyme, which was identified as the membrane‐type serine proteinase matriptase (MT‐SP1) by immunoblotting. When the membrane fraction was separated by SDS/PAGE, the IGFBP‐rP1‐cleaving activity comigrated with matriptase. A soluble form of matriptase purified in an inhibitor‐free form efficiently cleaved IGFBP‐rP1 at the same site as that found in a naturally cleaved IGFBP‐rP1. Furthermore, small interfering RNAs for matriptase efficiently blocked both the matriptase expression and the cleavage of IGBP‐rP1 in OVISE cells. These results demonstrate that IGFBP‐rP1 is processed to the two‐chain form by matriptase on the cell surface.

Production of soluble matriptase by human cancer cell lines and cell surface activation of its zymogen by trypsin

The membrane‐bound serine proteinase matriptase, which is often released from the plasma membrane of epithelial and carcinoma cells, has been implicated to play important roles in both physiological and pathological conditions. However, the regulatory mechanism of its activity is poorly understood. In the present study, we examined expression and activation state of soluble matriptase in 24 human cancer cell lines. Soluble matriptase was detected in the conditioned media from all of 5 colon and 4 breast carcinoma cell lines and 8 of 10 stomach carcinoma cell lines tested. Only two of five lung cancer cell lines released the matriptase protein into the culture media. Out of the five matriptase‐negative cell lines, two cell lines expressed the matriptase mRNA. Among 24 cancer cell lines tested, 13 cell lines secreted trypsin in an active or latent form and all of them released matriptase. Most of the 24 cell lines released a latent, single‐chain matriptase of 75 kDa as a major form, as well as low levels of complex forms of an activated two‐chain enzyme with its specific inhibitor HAI‐1. Thus, these soluble matriptases appeared to have little proteolytic activity. Treatment of stomach and colon cancer cell lines with epidermal growth factor stimulated the release of matripatase/HAI‐1 complexes. In cancer cell lines secreting active trypsin, however, matriptase was released mostly as an inhibitor‐free, two‐chain active form. Trypsin seemed to activate the membrane‐bound, latent matriptase on the cell surface. These results suggest that matriptase and trypsin cooperatively function for extracellular proteolysis.

Abstract B81: Angiomodulin (AGM/IGFBP-rP1) is overexpressed in tumor vasculature and regulates adhesion of vascular endothelial cells via integrin αvβ3: Possible roles in tumor angiogenesis

Angiomodulin (AGM) is a member of insulin-like growth factor binding protein (IGFBP) superfamily and often called IGFBP-rP1. Our past studies identified this protein as a tumor-derived cell adhesion factor (TAF) that is highly accumulated in blood vessels of human cancer tissues (PNAS 93:8384, 1996). Recently we found that AGM is overexpressed in not only vasculature but also stromal fibroblasts in some human cancers (Cancer Sci 4:691, 2012). AGM stimulates the growth of fibroblasts and their expression of fibronectin and α-smooth muscle actin, inducing their morphological change to a myofibroblast-like shape. It is supposed that AGM activates cancer-associated fibroblasts (CAFs) and increases cancer stroma. In contrast to these findings, tumor-suppressing activity of AGM has been reported by other studies. To clarify the roles of AGM in tumor progression, we here investigated the distribution of AGM in benign and invasive breast cancers and its regulatory factors and biological activities. Immunohistochemical analysis showed that AGM was greatly expressed in tumor vasculature even in ductal carcinoma in situ (DCIS) as compared to normal vasculature, while its expression in CAFs was more prominent in invasive carcinomas than DCIS. In vitro analyses showed that AGM was markedly induced by VEGF in vascular endothelial cells but by TGF-β in fibroblasts. Although AGM did not significantly affect the growth of endothelial cells, it potently promoted the adhesion of endothelial cells through integrins. Experiments with neutral anti-integrin antibodies revealed that the cell adhesion activity was mediated mainly by integrin αvβ3 and weakly by integrin α2β1. AGM induced cytoskeletal change in endothelial cells. Furthermore, AGM and integrin αvβ3 were colocalized at high levels in tumor vasculature. These results suggest that AGM regulates angiogenesis and other vascular functions as a ligand of integrin αvβ3 in cancer tissues. The data also indicate that AGM is induced by different mechanisms and plays different roles in tumor progression between CAFs and vasculature. Citation Format: Kaoru Miyazaki, Eriko Komiya, Hiroki Sato, Yohei Miyagi, Shouichi Higashi. Angiomodulin (AGM/IGFBP-rP1) is overexpressed in tumor vasculature and regulates adhesion of vascular endothelial cells via integrin αvβ3: Possible roles in tumor angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B81.