Vascular endothelial growth factor is an autocrine growth factor, signaling through neuropilin-1 in non-small cell lung cancer
Authors: Derek J Richard
Kenneth J O&#x27;Byrne
Emily Hams
Kathy Gately
Graham P Pidgeon
Anthony Mitchell Davies
Martin P Barr
Padraic G Fallon
Steven G Gray
Journal: Molecular Cancer (2015)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: VEGF pathway; lung cancer; Bevacizumab; NSCLC; tyrosine kinase inhibitors
Affiliations: 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. ., School of Medicine, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, Ireland. ., Irish National Centre for High Content Screening & Analysis, School of Clinical Medicine, Trinity College Dublin, Dublin, Ireland. ., Cancer & Ageing Research Program, Queensland University of Technology, Brisbane, Australia. ., Department of Surgery, Institute of Molecular Medicine, St James's Hospital & Trinity College Dublin, Dublin, Ireland. .

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. 

Retracted: No

PubPeer comments:
 Could the authors please check Figure 4? Several lanes in the Western blot representing A549 cells (in A) look unexpectedly similar to lanes representing SKMES1 cells (in B). Shown with green, pink, and orange boxes. Of note, the 3 lanes marked in pink appear to be visible twice within the same blot.  , 
 In Figure 1C, in both the  NP1 and  NP2 blots, the same areas appear to be visible multiple times within the same photo. Shown with red, blue, and mustard boxes.  , 
 The original blots in question will be located and examined to confirm the above, while excess protein lysates from these experiments will also be looked for should the B-actin need to be repeated for these samples in question. , 
 Unexpectedly, the journal issued a Correction on 27 January 2020, found here: https: molecular-cancer.biomedcentral.com articles 10.1186 s12943-020-1142-8  Since the publication of this work [1] and in response to a recent query that was brought to our attention in relation to the Western Blot in Figure 1(C) for NP2, protein lysates prepared around the same time as those presented in the manuscript in question, were run by SDS-PAGE under similar experimental conditions and probed using the same primary antibodies to NP1 and NP2 that were used originally. ,