title: Vascular endothelial growth factor is an autocrine growth factor, signaling through neuropilin-1 in non-small cell lung cancer.
abstract: BACKGROUND
  The VEGF pathway has become an important therapeutic target in lung cancer, where VEGF has long been established as a potent pro-angiogenic growth factor expressed by many types of tumors. While Bevacizumab (Avastin) has proven successful in increasing the objective tumor response rate and in prolonging progression and overall survival in patients with NSCLC, the survival benefit is however relatively short and the majority of patients eventually relapse. The current use of tyrosine kinase inhibitors alone and in combination with chemotherapy has been underwhelming, highlighting an urgent need for new targeted therapies. In this study, we examined the mechanisms of VEGF-mediated survival in NSCLC cells and the role of the Neuropilin receptors in this process.
  
  
  METHODS
  NSCLC cells were screened for expression of VEGF and its receptors. The effects of recombinant VEGF and its blockade on lung tumor cell proliferation and cell cycle were examined. Phosphorylation of Akt and Erk1/2 proteins was examined by high content analysis and confocal microscopy. The effects of silencing VEGF on cell proliferation and survival signaling were also assessed. A Neuropilin-1 stable-transfected cell line was generated. Cell growth characteristics in addition to pAkt and pErk1/2 signaling were studied in response to VEGF and its blockade. Tumor growth studies were carried out in nude mice following subcutaneous injection of NP1 over-expressing cells.
  
  
  RESULTS
  Inhibition of the VEGF pathway with anti-VEGF and anti-VEGFR-2 antibodies or siRNA to VEGF, NP1 and NP2 resulted in growth inhibition of NP1 positive tumor cell lines associated with down-regulation of PI3K and MAPK kinase signaling. Stable transfection of NP1 negative cells with NP1 induced proliferation in vitro, which was further enhanced by exogenous VEGF. In vivo, NP1 over-expressing cells significantly increased tumor growth in xenografts compared to controls.
  
  
  CONCLUSIONS
  Our data demonstrate that VEGF is an autocrine growth factor in NSCLC signaling, at least in part, through NP1. Targeting this VEGF receptor may offer potential as a novel therapeutic approach and also support the evaluation of the role of NP1 as a biomarker predicting sensitivity or resistance to VEGF and VEGFR-targeted therapies in the clinical arena.
authors.affiliation: Thoracic Oncology Research Group, School of Clinical Medicine, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital & Trinity College Dublin, Dublin, Ireland. mbarr@stjames.ie.
authors.forename: Martin P
authors.initials: MP
authors.lastname: Barr
authors.affiliation: Thoracic Oncology Research Group, School of Clinical Medicine, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital & Trinity College Dublin, Dublin, Ireland. sgray@stjames.ie.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Gray
authors.affiliation: Thoracic Oncology Research Group, School of Clinical Medicine, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital & Trinity College Dublin, Dublin, Ireland. kgately@stjames.ie.
authors.forename: Kathy
authors.initials: K
authors.lastname: Gately
authors.affiliation: School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland. hamse@tcd.ie.
authors.forename: Emily
authors.initials: E
authors.lastname: Hams
authors.affiliation: School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland. pfallon@tcd.ie.
authors.forename: Padraic G
authors.initials: PG
authors.lastname: Fallon
authors.affiliation: Irish National Centre for High Content Screening & Analysis, School of Clinical Medicine, Trinity College Dublin, Dublin, Ireland. anthonmitchelldavies@gmail.com.
authors.forename: Anthony Mitchell
authors.initials: AM
authors.lastname: Davies
authors.affiliation: Cancer & Ageing Research Program, Queensland University of Technology, Brisbane, Australia. derek.richard@qut.edu.au.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Richard
authors.affiliation: Department of Surgery, Institute of Molecular Medicine, St James's Hospital & Trinity College Dublin, Dublin, Ireland. pidgeong@tcd.ie.
authors.forename: Graham P
authors.initials: GP
authors.lastname: Pidgeon
authors.affiliation: Thoracic Oncology Research Group, School of Clinical Medicine, Institute of Molecular Medicine, Trinity Centre for Health Sciences, St. James's Hospital & Trinity College Dublin, Dublin, Ireland. k.obyrne@qut.edu.au.|Cancer & Ageing Research Program, Queensland University of Technology, Brisbane, Australia. k.obyrne@qut.edu.au.
authors.forename: Kenneth J
authors.initials: KJ
authors.lastname: O'Byrne
mesh_terms: D000818:Animals; D002097:C-Reactive Protein / Q000235:genetics*; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D015536:Down-Regulation / Q000235:genetics; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D020935:MAP Kinase Signaling System / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D040262:Receptors, Vascular Endothelial Growth Factor / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D000099153:Pentraxins
chemical_list: D002097:C-Reactive Protein; D020929:Mitogen-Activated Protein Kinase Kinases; D009419:Nerve Tissue Proteins; D019869:Phosphatidylinositol 3-Kinases; D040262:Receptors, Vascular Endothelial Growth Factor; D042461:Vascular Endothelial Growth Factor A; D000099153:Pentraxins; C467484:VEGFA protein, human