Tumor stroma marker endosialin (Tem1) is a binding partner of metastasis-related protein Mac-2 BP/90K

Renate Becker, Martin C. Lenter, Tobias Vollkommer, Anja M. Boos, Dennis Pfaff, Hellmut G. Augustin and Sven Christian

Tumor development involves complex bidirectional interactions between tumor cells and host stromal cells. Endosialin (Tem1) has been identified as a highly O-glycosylated transmembrane glycoprotein, which is specifically expressed by tumor vessel-associated pericytes and stromal fibroblasts of a wide range of human tumors. Recent experiments in endosialin-deficient mice have unraveled a critical role of endosialin in site-specific tumor progression and metastasis. To molecularly understand the mechanisms of endosialin function, we aimed to identify extracellular endosialin ligands and identified Mac-2 BP/90K as a specific interaction partner. Detailed biochemical analyses identified a C-terminal fragment of Mac-2 BP/90K, which was shown to contain binding sites for galectin-3, and collagens as the structures responsible for endosialin binding. Subsequent expression analysis of Mac-2 BP/90K in vivo revealed weak or no expression in most normal tissues and strong up-regulation in tumor cells of human neoplastic tissues. Intriguingly, the expression patterns of Mac-2 BP/90K and endosialin were mutually exclusive in all human tissues. Correspondingly, loss-of-function adhesion experiments of Mac-2 BP/90K-expressing tumor cells on endosialin-expressing fibroblasts revealed a repulsive outcome of the Mac-2 BP/90K interaction. Taken together, the experiments identify a novel repulsive interaction between endosialin on stromal fibroblasts and Mac-2 BP/90K on tumor cells.

Laminin deposition is dispensable for vasculogenesis but regulates blood vessel diameter independent of flow

Lars Jakobsson, Anna Domogatskaya, Karl Tryggvason, David Edgar, and Lena Claesson-Welsh

Basement membranes (BMs) consisting of laminins, collagens, and heparan sulfate proteoglycans (HSPGs) are vital for proper endothelial cell function, but many aspects of their role in vascular development remain unknown. Here, we demonstrate that vascular structures within differentiating embryoid bodies are wrapped in a BM composed of 4- and 5-chain laminins, fibronectin, collagen IV, and HSPGs. In sprouting angiogenesis, laminins were produced by stalk cells, as well as the leading tip cell, and deposited along the sprout length, including tip cell filopodia. In embryonic stem cells deficient in laminins, due to lamc1 (laminin 1) deletion, vascular development and organization were largely unaffected. However, the frequency of vessels with wide lumens was increased 4-fold. Laminin-deficient vessels were moreover characterized by increased fibronectin levels and enhanced endothelial cell proliferation. We conclude that laminins are dispensable for vascular development but that they regulate lumen formation in the absence of flow and vascular tone

Fibronectin Type I Repeat Is a Nonactivating Ligand for EphA1 and Inhibits ATF3-dependent Angiogenesis

Junko Masuda, Ryosuke Usui, and Yoshiro Maru

ATF3 stimulated promoter activity of EphA1 by 3.4-fold in ATF3-dependent angiogenesis in vitro. Although tyrosine kinase activation of EphA1 was dispensable, binding of EphA1 to fibronectin through its type I repeat played an essential role in the angiogenesis. Recombinant proteins containing fibronectin 10th to 12th type I repeat (I 10–12) but not I 12 could inhibit the angiogenesis in vitro by competitively targeting EphA1 with the full-length fibronectin. However, I 12 acquired a higher affinity toward EphA2 with Kd 18 nM and inhibited vascular endothelial growth factor-dependent angiogenic invasion in a Matrigel plug assay.

Matrix Metalloproteinase-activated Anthrax Lethal Toxin Demonstrates High Potency in Targeting Tumor Vasculature

Shihui Liu, Hailun Wang, Brooke M. Currie, Alfredo Molinolo, Howard J. Leung, Mahtab Moayeri, John R. Basile, Randall W. Alfano, J. Silvio Gutkind, Arthur E. Frankel, Thomas H. Bugge, and Stephen H. Leppla

Anthrax lethal toxin (LT), a virulence factor secreted by Bacillus anthracis, is selectively toxic to human melanomas with the BRAF V600E activating mutation because of its proteolytic activities toward the mitogen-activated protein kinase kinases (MEKs). To develop LT variants with lower in vivo toxicity and high tumor specificity, and therefore greater potential for clinical use, we generated a mutated LT that requires activation by matrix metalloproteinases (MMPs). This engineered toxin was less toxic than wild-type LT to mice because of the limited expression of MMPs by normal cells. Moreover, the systemically administered toxin produced greater anti-tumor effects than wild-type LT toward human xenografted tumors. This was shown to result from its greater bioavailability, a consequence of the limited uptake and clearance of the modified toxin by normal cells. Furthermore, the MMP-activated LT had very potent anti-tumor activity not only to human melanomas containing the BRAF mutation but also to other tumor types, including lung and colon carcinomas regardless of their BRAF status. Tumor histology and in vivo angiogenesis assays showed that this anti-tumor activity is due largely to the indirect targeting of tumor vasculature and angiogenic processes. Thus, even tumors genetically deficient in anthrax toxin receptors were still susceptible to the toxin therapy in vivo. Moreover, the modified toxin also displayed lower immunogenicity compared with the wild-type toxin. All these properties suggest that this MMP-activated anti-tumor toxin has potential for use in cancer therapy.

Extracellular matrix protein βig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation

Chaoyu Ma, Yu Rong, Daniel R. Radiloff, Michael B. Datto, Barbara Centeno, Shideng Bao, Anthony Wai Ming Cheng, Fumin Lin, Shibo Jiang, Timothy J. Yeatman, and Xiao-Fan Wang

Metastasis, the major cause of cancer death, is a multistep process that requires interactions between cancer cells and stromal cells and between cancer cells and extracellular matrix. Molecular alterations of the extracellular matrix in the tumor microenvironment have a considerable impact on the metastatic process during tumorigenesis. Here we report that elevated expression of βig-h3/TGFBI (transforming growth factor, β-induced), an extracellular matrix protein secreted by colon cancer cells, is associated with high-grade human colon cancers. Ectopic expression of the βig-h3 protein enhanced the aggressiveness and altered the metastatic properties of colon cancer cells in vivo. Inhibition of βig-h3 expression dramatically reduced metastasis. Mechanistically, βig-h3 appears to promote extravasation, a critical step in the metastatic dissemination of cancer cells, by inducing the dissociation of VE-cadherin junctions between endothelial cells via activation of the integrin vβ5–Src signaling pathway. Thus, cancers associated with overexpression of βig-h3 may have an increased metastatic potential, leading to poor prognosis in cancer patients.

Essential Role of Extracellular SOD in Reparative Neovascularization Induced by Hindlimb Ischemia

Ha Won Kim, Angela Lin, Robert E. Guldberg, Masuko Ushio-Fukai, Tohru Fukai

Neovascularization is an important physiological repair mechanism in response to ischemic injury, and its process is dependent on reactive oxygen species (ROS). Overproduction of superoxide anion (O2·–) rather contributes to various cardiovascular diseases. The extracellular superoxide dismutase (ecSOD) is one of the major antioxidant enzymes against O2·– in blood vessels; however, its role in neovascularization induced by tissue ischemia is unknown. Here we show that hindlimb ischemia of mice stimulates a significant increase in ecSOD activity in ischemic tissues where ecSOD protein is highly expressed at arterioles. In mice lacking ecSOD, ischemia-induced increase in blood flow recovery, collateral vessel formation, and capillary density are significantly inhibited. Impaired neovascularization in ecSOD–/– mice is associated with enhanced O2·– production, TUNEL-positive apoptotic cells and decreased levels of NO2–/NO3– and cGMP in ischemic tissues as compared with wild-type mice, and it is rescued by infusion of the SOD mimetic tempol. Recruitment of inflammatory cells into ischemic tissues as well as numbers of inflammatory cells and endothelial progenitor cells (c-kit+/CD31+ cells) in both peripheral blood and bone marrow (BM) are significantly reduced in these knockout mice. Of note, ecSOD expression is markedly increased in BM after ischemia. NO2–/NO3– and cGMP levels are decreased in ecSOD–/– BM. Transplantation of wild-type BM into ecSOD–/– mice rescues the defective neovascularization. Thus, ecSOD in BM and ischemic tissues induced by hindlimb ischemia may represent an important compensatory mechanism that blunts the overproduction of O2·–, which may contribute to reparative neovascularization in response to ischemic injury.