sists of four transmembrane receptors belonging to the receptor tyrosine kinase super family and includes EGFR, ErbB2/Neu/HER 2, ErbB3/HER 3, and ErbB4/HER 4. In prostate cancer, EGFR expression was detected in 18% of cancers CT99021 GSK-3 inhibitor and was significantly associated with high grade, advanced stage, and high risk for PSA recurrence in univariate analysis . EGFR is a transducer of the urokinase receptor initiated signal which is required for in vivo growth of a human carcinoma. uPAR, EGFR and integrins form a ternary complex which promotes cancer cell migration, invasion, proliferation and survival. Specific ligands such as uPA or EGF working through paracrine or autocrine loops are wellestablished activators of EGFR. In cells expressing very low levels of uPAR, which are dormant in vivo, the 51 integrin exists in an inactive state and associates poorly with EGFR.
In spite of its high expression, under both basal conditions or after cell adhesion to fibronectin, EGFR is not phosphorylated. In contrast, in cells expressing high levels of uPAR, this receptor, in the presence of uPA, interacts frequently with and activates 51, leading to the formation CP-690550 540737-29-9 of a multiprotein complex that contains FAK and EGFR, and that exhibits robust ERK activation. These results unveil a model whereby highly malignant human carcinoma cells, through overexpression of uPAR, are able to subvert and utilize a tightly regulated EGFR pathway to gain matrix derived proliferative advantage. High molecular weight kininogen is a multifunctional plasma protein that plays important roles in many pathophysiological processes, such as fibrinolysis, thrombosis, and inflammation.
Single chain HK consists of 6 domains and is complexed in plasma with prekallikrein. On the endothelial cell surface prekallikrein is cleaved by prolylcarboxpeptidase to kallikrein which releases bradykinin from domain 4 of HK to generate two chain high molecular weight kininogen. HKa undergoes extensive conformational changes to expose domain 5 and inhibits angiogenesis through these anti adhesive sites. HKa and D5 bind uPAR and induce apoptosis in endothelial cells by disrupting uPAR association with integrins v3 and 51 through cell detachment. uPAR mediates adhesion and signaling in endothelial cells by binding to vitronectin. D5 of HK binds the soluble uPAR receptor with 10 fold higher affinity than Domain 3.
Therefore, exposure of D5 in HKa is consistent with HKa having a higher affinity for uPAR than HK. In this study, we hypothesize that the binding of HKa and D5 to uPAR inhibits EGFR phosphorylation and would therefore inhibit tumor cell migration and invasion in prostate cancer. MATERIALS AND METHODS Reagents and Antibodies Two chain high molecular weight kininogen was purchased from Enzyme Research Laboratories. Collagen solution was purchased from BD Biosciences. Protease inhibitor cocktail was purchased from Sigma Co.. Antibodies directed against total and phosphorylationspecific Akt, total and phosphorylation specific extracellular signalregulated kinase were obtained from Cell Signaling Technology, Inc. Antibodies Liu et al. Page 2 Oncogene. Author manuscript, available in PMC 2010 April 28. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript against total and phosphorylation specific EGFR, polyclonal antibodies against integrin v and 1 were obtained from Santa Cruz Biotechno