Date Published: January 25, 2019
Publisher: Public Library of Science
Author(s): David Muench, Francine Rezzoug, Shelia D. Thomas, Jingjing Xiao, Ashraful Islam, Donald M. Miller, Kara C. Sedoris, Pier Giorgio Petronini.
Vascular endothelial growth factor (VEGF) is commonly overexpressed in a variety of tumor types including lung cancer. As a key regulator of angiogenesis, it promotes tumor survival, growth, and metastasis through the activation of the downstream protein kinase B (AKT) and extracellular signal-regulated kinase (ERK 1/2) activation. The VEGF promoter contains a 36 bp guanine-rich sequence (VEGFq) which is capable of forming quadruplex (four-stranded) DNA. This sequence has been implicated in the down-regulation of both basal and inducible VEGF expression and represents an ideal target for inhibition of VEGF expression.
Our experiments demonstrate sequence-specific interaction between a G-rich quadruplex-forming oligonucleotide encoding a portion of the VEGFq sequence and its double stranded target sequence, suggesting that this G-rich oligonucleotide binds specifically to its complementary C-rich sequence in the genomic VEGF promoter by strand invasion. We show that treatment of A549 non-small lung cancer cells (NSCLC) with this oligonucleotide results in decreased VEGF expression and growth inhibition. The VEGFq oligonucleotide inhibits proliferation and invasion by decreasing VEGF mRNA/protein expression and subsequent ERK 1/2 and AKT activation. Furthermore, the VEGFq oligonucleotide is abundantly taken into cells, localized in the cytoplasm/nucleus, inherently stable in serum and intracellularly, and has no effect on non-transformed cells. Suppression of VEGF expression induces cytoplasmic accumulation of autophagic vacuoles and increased expression of LC3B, suggesting that VEGFq may induce autophagic cell death.
Our data strongly suggest that the G-rich VEGFq oligonucleotide binds specifically to the C-rich strand of the genomic VEGF promoter, via strand invasion, stabilizing the quadruplex structure formed by the genomic G-rich sequence, resulting in transcriptional inhibition. Strand invading oligonucleotides represent a new approach to specifically inhibit VEGF expression that avoids many of the problems which have plagued the therapeutic use of oligonucleotides. This is a novel approach to specific inhibition of gene expression.
Vascular Endothelial Growth Factor (VEGF) plays a key role in tumor cell growth; causing increased proliferation, angiogenesis, and metastasis in a variety of tumor types including lung cancer.[1, 2] Expression of VEGF is primarily regulated at the transcriptional level and its expression can be induced physiologically by tumor hypoxia, hypoglycemia, loss of tumor suppressor genes, or by activation of growth factor signaling cascades.[3–8] Clinical studies have correlated increased VEGF mRNA and protein levels with tumor progression, leading to poorer prognosis and post-operative outcome in both NSLC and small cell lung cancer.[9–12] Binding of VEGF to its receptor stimulates the downstream kinases, ERK and AKT, driving proliferation, angiogenesis, cell invasion/migration, and cell survival, processes which are critical for lung tumor survival, growth, and metastasis. Thus, reduction of VEGF expression could reasonably be expected to attenuate tumor growth and to represent a potential anti-cancer approach.
The promoters of many cancer-related genes, including VEGF, contain sequences that are capable of forming intramolecular quadruplex (four-stranded) DNA structures . These G-rich sequences are generally located within nuclease hypersensitivity regions which play important roles in regulating expression of their downstream genes. Using quadruplex-specific antibodies, Balasubrumanian has shown that quadruplex formation occurs in intact cells [15, 16] and that quadruplex structures are disproportionately represented in regulatory chromatin . Previous work has suggested that the quadruplex-forming sequence within the VEGF promoter plays an important role in downregulation of basal and inducible VEGF transcription [8,19,24]. This is consistent with the transcriptional inhibitory activity of quadruplex-forming sequences in other promoters, including the c-MYC  and hTERT  promoters. In fact, mutations in the hTERT quadruplex-forming sequence are seen quite commonly in melanoma [36, 37] and glioblastoma,  and less commonly in other tumors, as well. These mutations have been shown to destabilize the hTERT promoter quadruplex structure and, presumably, abrogate the transcriptional inhibitory activity which occurs after quadruplex formation. 
Our studies demonstrate that treatment of NSCLC cells with oligonucleotides encoding the VEGFq sequence inhibits cell growth and attenuates cell invasion through a mechanism involving inhibition of VEGF expression, downregulation of activated ERK 1/2 and AKT, and eventually induction of cell death. The demonstration of sequence-specific binding of the VEGFq oligonucleotide to its double stranded target sequence strongly supports the hypothesis that VEGFq binds to the genomic VEGF promoter by strand invasion in a sequence-specific (Watson-Crick) manner. This would, of necessity, stabilize the quadruplex structure formed by the G-rich strand of the genomic VEGF promoter, resulting in transcriptional inhibition of VEGF expression. It is evident that the cellular effects of VEGFq are quite different from those of Pu27, further supporting the gene-specificity of these oligonucleotides. This study suggests that the VEGFq oligonucleotide may represent a novel therapy for the treatment of NSCLC alone or in combination with standard lung cancer chemotherapies. This approach of gene-specific inhibition of transcription can be applied to the wide array of genes whose promoters contain quadruplex-forming sequences.