Use of siRNAs and Antisense Oligonucleotides Against Survivin RNA to Inhibit Steps Leading to Tumor Angiogenesis

Abstract

The antiapoptotic protein survivin is an attractive target in cancer therapy because it is expressed differently in tumors and normal tissues and it is potentially required for cancer cells to remain viable. Given that survivin is also overexpressed in endothelial cells (ECs) of newly formed blood vessels found in tumors, its RNA targeting might compromise EC viability and interfere with tumor angiogenesis. We used two antisense strategies against survivin expression, antisense oligonucleotides (aODN) and small interfering RNA (siRNA), to study in ECs the contribution of survivin in various steps leading to tumor angiogenesis. A 21-mer phosphorothioate aODN and two siRNA oligonucleotides against survivin mRNA were designed to downregulate survivin expression. Survivin targeting caused (1) a strong growth-inhibitory effect, (2) a 4-fold increase in apoptosis, (3) an accumulation of cells in the S phase and a decrease in G2/M phase, (4) a dose-dependent inhibition of EC migration on Vitronectin, and (5) a decrease in capillary formation. Control oligonucleotides, an unrelated oligonucleotide, and one with four mismatches, had no significant effect. All these results show that survivin is a suitable target in cancer therapy because its inhibition in EC causes both a proapoptotic effect and an interruption of tumor angiogenesis. The two strategies used, classic aODN and siRNA technology, were very effective. Moreover, the latter can be used in the low nanomolar range, thus increasing the sensitivity of the treatment.

Dissecting the role of endothelial SURVIVIN ΔEx3 in angiogenesis

“…No significant changes in proliferation were observed, suggesting Survivin ⌬Ex3 does not play a significant role in regulating cell growth ( Figure 4C). Treatment of HUVE cells with a combination of shRNAs that target all Survivin splice variants, with the exception of Survivin 2␣, resulted in a statistically significant increase in apoptosis (mean ϭ 32.5%), as has been previously demonstrated 26 ( Figure 4A). …”

“…Studies of the Survivin splice variants have been performed almost exclusively with cancer cells; however, Survivin itself has been increasingly implicated in other physiologic and pathophysiologic processes, including angiogenesis. [22][23][24][25][26] Previous studies have shown that the main isoform of Survivin is expressed at higher levels in the endothelial cells of newly formed blood vessels within tumors 33 as well as within injured or ischemic brain. 34 Functional studies have not yet addressed the roles of the individual splice variants in angiogenesis.…”

“…34 One report using antisense oligonucleotides and siRNA to Survivin in endothelial cells showed that down-regulation of Survivin led to growth inhibition, increased apoptosis, and decreased capillary formation. 26 However, in this work, based on the siRNA design, all Survivin isoforms would have been targeted, eliminating the possibility of evaluating the contribution of the individual Survivin splice variants. Disruption of Survivin using antisense and dominant-negative mutants that affect all of the Survivin variants resulted in a decrease in new tumor formation and in tumor angiogenesis.…”

“…[22][23][24][25] Down-regulation of Survivin by antisense oligonucleotides and siRNAs results in an increase in apoptosis in these cells. 26 In this work, we examined the effects of Survivin ⌬Ex3 on apoptosis and proliferation in vascular endothelial cells by treatment with a Survivin ⌬Ex3-specific shRNA, targeting the exon 2-exon 4 boundary, which does not target any other isoform except Survivin ⌬Ex3 ( Figure 5A). Treatment with this shRNA resulted in a 30% to 50% reduction of Survivin ⌬Ex3 transcripts, as assayed by Taqman real-time reverse-transcription (RT)-PCR (not shown), and an appreciable reduction in Survivin ⌬Ex3 protein ( Figure S5).…”

Impacts of survivin and caspase-3 on apoptosis and angiogenesis in oral cancer

“…Survivin may also promote the proliferation of endothelial cells, and silencing survivin may be able to inhibit the expression of VEGF (25). The import of survivin-targeting antisense oligonucleotides into cultured endothelial cells may promote the apoptosis of these endothelial cells and inhibit angiogenesis (26,27). A previous study has demonstrated that basic fibroblast growth factor and survivin also produce synergistic effects, with positively associated expression patterns in lung cancer and OSCC (23).…”