Abstract 4996: The VEGF quadruplex-forming sequence inhibits lung cancer cell growth

Abstract

Abstract Vascular endothelial growth factor (VEGF), commonly overexpressed in a variety of malignancies including lung cancer, is a key regulator of angiogenesis promoting tumor survival, growth, and metastasis. The promoters of several cancer-related genes, including VEGF, contain disproportionate sequences within nuclease hypersensitivity regions capable of forming quadruplex (four-stranded) DNA. The VEGF quadruplex-forming sequence (VEGF q), a polyG/polyC tract located in the proximal promoter region upstream of the transcription initiation site, has been implicated in the regulation of both basal and inducible VEGF expression. However, the biological role of this sequence remains unclear. We hypothesize that treatment of A549 non-small lung cancer cells with an oligonucleotide encoding VEGF q inhibits cell growth by decreasing VEGF expression and subsequent signaling through VEGF receptor 2 (FLK-1). To determine the biological role of VEGF on lung cancer cell proliferation, A549 cells were treated with VEGF q or the corresponding mutant sequence (MutVEGF), which lacks runs of two or more guanines. Our results demonstrate that VEGF q formed a parallel quadruplex structure and showed remarkable serum and intracellular stability. Confocal and flow cytometry analysis of cells treated with FITC-labeled VEGF q showed prominent uptake and nuclear/cytoplasmic localization in contrast to MutVEGF. Treatment of A549 cells with VEGF q caused a significant dose and time-dependent decrease in cell proliferation after 3 and 6 days (IC50<5µM) as determined by MTT assay, which was not a result of cell cycle arrest. Matrigel analysis also showed a significant reduction in tumor invasiveness after 24 h with VEGF q treatment. Although uptake of VEGF q occurred in non-transformed Hs27 human fibroblast cells, no change in cell growth was evident, indicating that the anti-proliferative effects of VEGF q are specific for malignant cells. Inhibition of cell proliferation and invasion corresponded with decreased VEGF protein expression resulting in decreased FLK-1 signaling and phosphorylation of the target proteins ERK 1/2 and AKT. These results demonstrate striking growth inhibition and decreased invasion of A549 cells in response to VEGF q related to inhibition of VEGF expression and signaling through FLK-1 and have significant therapeutic implications for the treatment of non-small cell lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4996. doi:10.1158/1538-7445.AM2011-4996