The Eph family of receptors is the largest family of RTKs. Eph receptors are stimulated by a family of membrane-linked ligands designated ephrins (6, 7). Both biochemical and genetic studies have established the central role that ephrins have in the control of cell contact repulsion, boundary formation, cell migration, and repulsive axon guidance (6). Repulsive axon guidance appears to be caused by modulation of cytoskeletal organization leading to regulation of neural growth-cone development (8). Eph-receptors also regulate cell-matrix interaction and cell proliferation by affecting signaling by integrins (9-11) and by modulation of MAPK response (12)(13)(14).In this article, we demonstrate that EphA4 binds directly and specifically via the N-terminal portion of its protein tyrosine kinase core to the juxtamembrane (JM) region of FGFRs. In cells that express EphA4 and FGFRs, the interactions between the cytoplasmic domains of EphA4 and FGFRs can lead to transreceptor activation, resulting in tyrosine phosphorylation of FRS2␣ and MAPK activation. The synergistic effect of ephrin-A1 stimulation on FGF2-induced cellular responses may influence the biological outcome of the activation of these two families of RTKs. Materials and MethodsYeast Two-Hybrid Experiments. The yeast two-hybrid system was used as described (15). The bait used for screening was 81 aa (amino acids 398-478), derived from the JM domain of human FGFR3. A human brain cDNA library in a pJG4-5 vector consisting of 3.5 ϫ 10 6 primary transformants (Clontech) was used for screening for proteins that interact with the JM domain of FGFR3. Fig. 1A shows the constructs used to detect interactions between the cytoplasmic domain of EphA4 and the JM domain of FGFR3.Cells. HEK293 cells were maintained in DMEM supplemented with 10% calf serum. For neural differentiation, P19 cells were maintained in ␣-MEM supplemented with 10% FBS containing 0.5 M retinoic acid for 3 days. Rat L6 myoblasts were maintained in DMEM supplemented with 10% FBS.Preparation of Ephrin-A1. Ephrin-A1 fused to human IgG-Fc was purchased from Sigma-Aldrich. Before application to the cells, 5 g of ephrin-A1-Fc was oligomerized by mixing with 12 g of rabbit anti-human IgG-Fc (Jackson ImmunoResearch) in 1 ml of PBS at 4°C for at least 1 h. As a control, a human IgG-Fc fragment (Jackson ImmunoResearch) was also applied after oligomerization.Expression Plasmids. Full-length cDNA of human EphA4 was prepared by RT-PCR using total RNA from a human brain extract (Clontech) as the template. The cDNA of human FGFR4 was prepared by RT-PCR using K562 cell-derived RNA as the template. The cDNAs for FGFR1 and FGFR2 were provided by W. McKeehan (Texas A&M University, College Station, TX). The cDNA for FGFR3 was provided by D. E. Johnson (University of Pittsburgh, Pittsburgh). Receptor mutants were prepared by applyConflict of interest statement: No conflicts declared.

The Eph receptor tyrosine kinases and their ephrin ligands form a unique cell-cell contact-mediated bidirectional signaling mechanism for regulating cell localization and organization. High expression of Eph receptors in a wide variety of human tumors indicates some roles in tumor progression, which makes these proteins potential targets for anticancer therapy. For this purpose, we did gene expression profiling for 47 surgical specimens of brain tumors including 32 high-grade glioma using a microarray technique. The analysis, focused on the receptor tyrosine kinases, showed that EphA4 mRNA in the tumors was 4-fold higher than in normal brain tissue. To investigate the biological significance of EphA4 overexpression in these tumors, we analyzed EphA4-induced phenotypic changes and the signaling mechanisms using human glioma U251 cells. EphA4 promoted fibroblast growth factor 2-mediated cell proliferation and migration accompanied with enhancement of fibroblast growth factor 2-triggered mitogen-activated protein kinase and Akt phosphorylation. In addition, active forms of Rac1 and Cdc42 increased in the EphA4-overexpressing cells. Furthermore, we found that EphA4 formed a heteroreceptor complex with fibroblast growth factor receptor 1 (FGFR1) in the cells and that the EphA4-FGFR1 complex potentiated FGFR-mediated downstream signaling. Thus, our results indicate that EphA4 plays an important role in malignant phenotypes of glioblastoma by enhancing cell proliferation and migration through accelerating a canonical FGFR signaling pathway. [Mol Cancer Ther 2008;7(9):2768 -78]

SRPX2 (Sushi repeat containing protein, X-linked 2) was first identified as a downstream molecule of the E2A-HLF fusion gene in t(17;19)-positive leukemia cells and the biological function of this gene remains unknown. We found that SRPX2 is overexpressed in gastric cancer and the expression and clinical features showed that high mRNA expression levels were observed in patients with unfavorable outcomes using real-time RT-PCR. The cellular distribution of SRPX2 protein showed the secretion of SRPX2 into extracellular regions and its localization in the cytoplasm. The introduction of the SRPX2 gene into HEK293 cells did not modulate the cellular proliferative activity but did enhance the cellular migration activity, as shown using migration and scratch assays. The conditioned-medium obtained from SRPX2-overexpressing cells increased the cellular migration activity of a gastric cancer cell line, SNU-16. In addition, SRPX2 protein remarkably enhanced the cellular adhesion of SNU-16 and HSC-39 and increased the phosphorylation levels of focal adhesion kinase (FAK), as shown using western blotting, suggesting that SRPX2 enhances cellular migration and adhesion through FAK signaling. In conclusion, the overexpression of SRPX2 enhances cellular migration and adhesion in gastric cancer cells. Here, we report that the biological functions of SRPX2 include cellular migration and adhesion to cancer cells.

Purpose: AZD2171 is an oral, highly potent, and selective vascular endothelial growth factor signaling inhibitor that inhibits all vascular endothelial growth factor receptor tyrosine kinases. The purpose of this study was to investigate the activity of AZD2171in gastric cancer. Experimental Design: We examined the antitumor effect of AZD2171 on the eight gastric cancer cell lines in vitro and in vivo. Results: AZD2171 directly inhibited the growth of two gastric cancer cell lines (KATO-III and OCUM2M), with an IC 50 of 0.15 and 0.37 Amol/L, respectively, more potently than the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib. Reverse transcription-PCR experiments and immunoblotting revealed that sensitive cell lines dominantly expressed COOH terminust runcated fibroblast growth factor receptor 2 (FGFR2) splicing variants that were constitutively phosphorylated and spontaneously dimerized. AZD2171 completely inhibited the phosphorylation of FGFR2 and downstream signaling proteins (FRS2, AKT, and mitogen-activated protein kinase) in sensitive cell lines at a 10-fold lower concentration (0