The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase-1, Flk-1) tyrosine kinase receptor is essential for VEGF-mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co-coordinated in response to VEGF-A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF-A dependent on constitutive endocytosis coordinated with ligand-activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand-stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs)/signal-transducing adaptor molecule (STAM) complex in a ligand and time-dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF-A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin-linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand-activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF-A-mediated response within the vascular system.

Protein kinase D (PKD) family members are increasingly implicated in multiple normal and abnormal biological functions, including signaling pathways that promote mitogenesis in pancreatic cancer. However, nothing is known about the effects of targeting PKD in pancreatic cancer. Our PKD inhibitor discovery program identified CRT0066101 as a specific inhibitor of all PKD isoforms. The aim of our study was to determine the effects of CRT0066101 in pancreatic cancer. Initially, we showed that autophosphorylated PKD1 and PKD2 (activated PKD1/2) are significantly upregulated in pancreatic cancer and that PKD1/2 are expressed in multiple pancreatic cancer cell lines. Using Panc-1 as a model system, we showed that CRT0066101 reduced bromodeoxyuridine incorporation; increased apoptosis; blocked neurotensin-induced PKD1/2 activation; reduced neurotensin-induced, PKD-mediated Hsp27 phosphorylation; attenuated PKD1-mediated NF-κB activation; and abrogated the expression of NF-κB-dependent proliferative and prosurvival proteins. We showed that CRT0066101 given orally (80 mg/kg/d) for 24 days significantly abrogated pancreatic cancer growth in Panc-1 subcutaneous xenograft model. Activated PKD1/2 expression in the treated tumor explants was significantly inhibited with peak tumor concentration (12 μmol/L) of CRT0066101 achieved within 2 hours after oral administration. Further, we showed that CRT0066101 given orally (80 mg/kg/d) for 21 days in Panc-1 orthotopic model potently blocked tumor growth in vivo. CRT0066101 significantly reduced Ki-67-positive proliferation index (P < 0.01), increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (P < 0.05), and abrogated the expression of NF-κB-dependent proteins including cyclin D1, survivin, and cIAP-1. Our results show for the first time that a PKD-specific small-molecule inhibitor CRT0066101 blocks pancreatic cancer growth in vivo and show that PKD is a novel therapeutic target in pancreatic cancer. Mol Cancer Ther; 9(5); 1136-46. ©2010 AACR.

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A 165 (VEGF-A

BACKGROUND: Neuropilin-1 (NRP1) is a non-tyrosine kinase receptor for vascular endothelial growth factor (VEGF) recently implicated in tumour functions. METHODS: In this study we used a specific antagonist of VEGF binding to the NRP1 b1 domain, EG3287, to investigate the functional roles of NRP1 in human carcinoma cell lines, non-small-cell lung A549, kidney ACHN, and prostate DU145 cells expressing NRP1, and the underlying mechanisms involved. RESULTS: EG3287 potently displaced the specific binding of VEGF to NRP1 in carcinoma cell lines and significantly inhibited the migration of A549 and ACHN cells. Neuropilin-1 downregulation by siRNA also decreased cell migration. EG3287 reduced the adhesion of A549 and ACHN cells to extracellular matrix (ECM), and enhanced the anti-adhesive effects of a b1-integrin functionblocking antibody. EG3287 increased the cytotoxic effects of the chemotherapeutic agents 5-FU, paclitaxel, or cisplatin on A549 and DU145 cells, through inhibition of integrin-dependent cell interaction with the ECM. CONCLUSIONS: These findings indicate that NRP1 is important for tumour cell migration and adhesion, and that NRP1 antagonism enhances chemosensitivity, at least in part, by interfering with integrin-dependent survival pathways. A major implication of this study is that therapeutic strategies targeting NRP1 in tumour cells may be particularly useful in combination with other drugs for combating tumour survival, growth, and metastatic spread independently of an antiangiogenic effect of blocking NRP1.

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