Discoidin domain receptors (DDRs), consisting of two closely related receptor types I and II, were originally found as an orphan receptor tyrosine kinase overexpressed in cancer cells.1,2) Later various types of collagens were identified as their activating ligand, indicating that they function to transmit signals from the extracellular matrix to inside the cells. 3,4) Five isoforms of DDR1 have been reported, which have differences in the cytoplasmic domain generated by alternative splicing, whereas DDR2 showed a single form. 5)Collagen type I, II, III, IV and VIII activate DDR1, whereas DDR2 is activated by type I, II, III and X. 3,4,6) The N-terminal extracellular domain of DDRs contains a stretch of about 150 amino acids, termed the discoidin domain, 1) which is critical for their binding to collagens. [7][8][9] The cytosolic domain of DDRs contains a tyrosine kinase motif whereby it is auto-phosphorylated, by activation with collagen, to transduce signals. 3,4) Activated signaling of DDR2 promotes fibroblast cell growth and migration through the induction of matrix metalloprotease (MMP) proteins. 10) As DDR2 has been linked to regulation of cell growth, its enhanced expression was observed in various human pathogenesis. Increased mRNA of DDR2 was found in classical Hodgkin's Lymphoma cells, 11) nasopharyngeal carcinoma 12) and correlated with the invasive property of mammary carcinoma cell lines.13) Elevated tyrosine phosphorylation of DDR2 was frequently detected in tumor samples of non small cell lung carcinoma.14)The increased synthesis and continuous activation of DDR2 was responsible for the over-proliferation of stellate cells and deregulated accumulation of fiber collagens in liver cirrhosis.15) High expression levels of DDR2 were detected in chondrocytes of an osteoathritic mouse model, which resulted in the upregulation of matrix metalloprotease-13 (MMP-13). MMP-13 is one of the main proteases that degrades collagen within the cartilage of osteoathritic patients.16) Synovial fibroblast isolated from the joint region of rheumatoid arthritis patients was reported to express a high level of DDR2, which then induces the synthesis of a large amount of matrix metalloprotease-1 that causes a serious damage on the cartilage.17) These studies imply that inhibition of DDR2 signaling could be a therapeutic approach and the development of inhibitors to prevent the activation of DDR2 by its ligand could be a useful strategy for the therapeutic agents.Actinomycin D is an antibiotic compound that has been clinically used for a long time as an anticancer drug to treat wilms' tumor, rhabdomyosarcoma, Ewing's sarcoma, trophoblastic neoplasia and testicular carcinoma. 18,19) The anticancer mechanism of actinomycin D has been thought to be mediated by its activity to intercalate into DNA and to inhibit RNA synthesis by binding to guanine residues and inhibiting DNA-dependent RNA polymerase. 20)In this paper, we found a new biological function of actinomycin D to interfere the activation of DDR2 by its ligand collagen...

-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.