Survivin expression promotes VEGF-induced tumor angiogenesis via PI3K/Akt enhanced β-catenin/Tcf-Lef dependent transcription.

Jaime G Fernández,Diego A Rodríguez,Carlos Rosas,Claudia Calderon,Julio C Tapia,Andrew Fg Quest,Lisette Leyton,Manuel Valenzuela,David Lemus,Ulises Urzúa,Natalia Díaz,Mathew C Wright,David Munroe

doi:10.1186/1476-4598-13-209

Abstract

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-209) contains supplementary material, which is available to authorized users.

Highlights

Angiogenesis is a physiological process characterized by the generation of new blood vessels from preexisting ones
(See figure on previous page.) Figure 1 Survivin expression increased endogenous survivin and β-catenin protein levels, as well as β-catenin/Tcf-Lef dependent transcriptional activity: A,B: HEK293T cells (5×105) were transfected with pEGFP-C1 or pEGFP-survivin (A), pcDNA or pcDNA-survivin (B) or treated with SB216763 (20 μM), a pharmacological inhibitor of Glycogen Synthase Kinase 3β (GSK-3β). β-catenin, survivin and actin protein levels were evaluated by western blotting
F-H: cells were cotransfected with pTOP-FLASH or pFOP-FLASH (1 μg) vectors and luciferase activity was obtained by calculating the pTOP-FLASH/pFOP-FLASH activity ratios for each condition

Summary

Introduction

Angiogenesis is a physiological process characterized by the generation of new blood vessels from preexisting ones. The inhibitor of apoptosis protein (IAP) survivin has been ascribed highly pleiotropic functions and is associated with tumor progression, metastasis and angiogenesis [4]. Survivin is overexpressed in essentially all human cancers and generally absent in normal adult tissues [5]. As an IAP, this protein is implicated in the inhibition of apoptosis, the mechanism by which this is achieved remains a matter of debate. Some possibilities include interaction and stabilization of the anti-apoptotic proteins XIAP [7] or HBXIP [8] and inhibition of pro-apoptotic proteins like second mitochondria-derived activator of caspases/ direct inhibitor of apoptosis binding protein with low pI (SMAC/DIABLO) [9] or Apoptosis Inducing Factor (AIF) [10]. More recently survivin has been shown to promote invasion and metastasis by enhancing Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB)dependent transcription of fibronectin [11]

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Discussion

Conclusion