Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

Chryso Kanthou,Sofia A Dos Santos,Sheila Harris,Gillian M Tozer,Diane V Lefley,Andrew J Steele,Claudia Coralli-Foxon,Olga Greco,Gabi U Dachs,William R English,Constantino C Reyes-Aldasoro

doi:10.1371/journal.pone.0104015

Abstract

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions.

Highlights

Vascular endothelial growth factor-A (VEGF) plays a fundamental role in tumour growth, vascularisation and metastasis and exists as multiple isoforms derived by alternative splicing of the VEGF gene [1]
In which VEGF expression is maintained under the control of its endogenous physiological promoter, we have discovered that individual isoforms are associated with distinct roles in terms of tumour cell behaviour and gained insight into signalling pathways that are linked with these effects
We demonstrate that endogenous single VEGF isoform expression is linked to distinct growth, survival and migratory characteristics in Ras transformed fibrosarcoma cells

Summary

Introduction

Vascular endothelial growth factor-A (VEGF) plays a fundamental role in tumour growth, vascularisation and metastasis and exists as multiple isoforms derived by alternative splicing of the VEGF gene [1]. Mouse and human proteins of 120/121, 164/165 and 188/189 amino acids respectively, represent major VEGF splice variants with distinct properties and expression patterns. These isoforms differ in terms of binding affinities to the extracellular matrix and receptor activation. Tumours display highly variable levels of relative isoform expression, with VEGF164/165 and VEGF120/121 generally being the most predominant and VEGF-188/189 relatively less abundant [2]. The different affinities to matrix, displayed by the various VEGF splice variants generate gradients in vivo and result in different signalling responses, which are important for angiogenesis [4,5]. VEGF has complex functions in angiogenesis-independent aspects of tumour growth and tumour cells have been shown to express functional VEGF receptors [6,7,8] but the role of individual VEGF isoforms in these processes remains poorly understood

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Conclusion