Abstract 5254: Cedarinib inhibits VEGF-stimulated proliferation in NSCLC cell lines.
Authors: Devery Aoife M. , Wadeka Rekha , Bokobza Sivan M. , Weber Anika M. , Jiang Yanyan , Ryan Anderson J. 
Published: August 2014

Abstract
Background: Vascular endothelial growth factor (VEGF) is a key stimulator of physiological and pathological angiogenesis. VEGF signals primarily through VEGF receptor 2 (VEGFR2), a receptor tyrosine kinase whose expression is generally considered to be restricted to endothelial cells. Recently, tumor cell VEGFR2 expression has been reported in several tumor settings, including non-small cell lung cancer (NSCLC). The purpose of this study was to determine the role of VEGFR2 in NSCLC cells, and to investigate the impact of inhibiting VEGFR2 signaling.
Methods: NSCLC tissue sections (n=52) were stained for VEGFR2 and CD31 expression by immunohistochemistry (IHC). Total and phosphorylated VEGFR2, AKT and MAPK protein levels in human NSCLC cell lines (n=8) were determined by Western blotting. VEGFR2 mRNA levels were measured by RT-PCR. For cell proliferation assays, cells were seeded (4x10ˆ3 per well) and 24 h later treated with VEGF (0-100 ng/mL) +/- cediranib (a potent and selective inhibitor of VEGF signaling). Alternatively, cells were treated with VEGFR2 siRNA 24 h post plating (4x10ˆ3 per well) and then treated with VEGF (0-100 ng/mL). Cell growth was quantified 5 d after VEGF treatment using crystal violet staining. For cytotoxicity assays, cells (2x10ˆ4 per well) were plated and 24 h later treated with VEGF, chemotherapy agents or cedarinib and incubated for 48 h. For radiation, cells were exposed to 137Cs (10 Gy, 1.958 Gy/min) 4 h after VEGF treatment. 48 h after VEGF treatment Hoechst 33258 (25 μM, 30 min) was added and apoptotic cells quantified by fluorescence (INCELL Analyzer 1000).
Results: IHC analysis of tissue sections demonstrated VEGFR2 expression in both adenocarcinoma and squamous cell carcinoma, and in endothelial cells. Western blot analysis showed expression of VEGFR2 protein in 3/8 NSCLC cell lines that correlated with VEGFR2 mRNA expression levels. VEGF-dependent VEGFR2 activation (increased pVEGFR2 and pMAPK but not pAKT) was apparent in NSCLC cells, and was associated with increased proliferation (1.4 - 1.9 fold, p&lt;0.05) in 2/3 of the VEGFR2-expressing NSCLC cell lines. Cedarinib inhibited both VEGF-dependent increases in cell proliferation and VEGFR2 downstream signaling. Knock down of VEGFR2 protein expression using siRNA also reduced VEGF-stimulated NSCLC cell proliferation. Inhibition of VEGFR2 tyrosine kinase activity using cediranib was more effective than inhibition of AKT (MK2206) or MEK (AZD6244) for overcoming VEGFR2-driven cell proliferation. VEGF treatment did not affect cell survival following treatment with radiation, cisplatin, docetaxel or gemcitabine.
Conclusion: Our data suggest that a subset of NSCLC patients may express functional tumor cell VEGFR2 which can act to promote VEGF-dependent tumor cell growth. In this patient subset, therapies targeting VEGFR2 signaling, such as cedarinib, may have the potential to inhibit both tumor cell proliferation and angiogenesis.
Citation Format: Aoife M. Devery, Rekha Wadeka, Sivan M. Bokobza, Anika M. Weber, Yanyan Jiang, Anderson J. Ryan. Cedarinib inhibits VEGF-stimulated proliferation in NSCLC cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5254. doi:10.1158/1538-7445.AM2013-5254