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

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

Migration of fibrosarcoma cells expressing single VEGF isoforms.

<p><b>a</b>): Wound closure was measured at intervals up to 24 h. Each point represents data obtained from 2–5 independent experiments ± SEM; <b>b</b>): representative culture images of the four fibrosarcoma cell lines showing wound closure at 18 h.</p

Fibrosarcoma cell adhesion and spreading.

<p><b>a</b>): Cell adhesion to uncoated plastic or plastic coated with collagen I, fibronectin or laminin-1 at 45 min. * and ** represent significant differences, (<b>*</b>p<0.05 and **p<0.01) by two way ANOVA followed by Bonferroni post-test. <b>b</b>): Cell spreading on fibronectin and laminin at 30 min, on a thin layer of collagen at 3 h or on collagen for 3 h in the presence of 2% FCS (collagen+S). Cells were stained live with with CellMask orange. Scale bars, 50 µm. <b>c</b>): Immunoblot analysis for β1 integrin (β-Itg) ILK and N-cadherin. Blots were normalised to actin. Results are representative of at least 3 independent experiments.</p

Schematic of proposed signalling interactions and morphological and motility characteristics of fibrosarcomas expressing single VEGF isoforms.

<p>Differences in PI3K/AKT, Ras/Raf/MEK/ERK and JAK/Stat3 signalling between fswt/fs188 and fs164/fs120 cells are shown. Upregulation/downregulation of activities or levels of expression of individual proteins are indicated by up or down arrows. VEGF188 expression is associated with reduced proliferation rates, increased apoptosis and typical integrin-dependent mesenchymal morphology and migration mode. VEGF164/VEGF120 expression is associated with faster proliferation rates, increased survival and rounded/amoeboid integrin-independent morphology and mode of motility. Migration of fs164/fs120 but not fswt/fs188 cells is contractility-dependent.</p

Signalling pathway analysis in fibrosarcomas expressing single VEGF isoforms.

<p><b>a–d</b>): Where indicated cells were incubated overnight with JAK inhibitor P6 (5 µM), or PI3K inhibitor LY20990 (10 µM). Equal amounts of proteins (10–30 µg) were analysed for <b>a</b>): phospho-AKT (p-AKT) and p70S6; <b>b</b>): pGSK3, pFOXO1, p27 and p21; <b>c</b>): phospho-ERKs1/2 (p-ERK) and total ERK (t-ERK); <b>d</b>): phospho-Stat3 (p-Stat3). <b>e</b>): Proteins extracted from solid tumours (50 µg/lane) and analysed for pStat3. All blots were normalized with actin or GAPDH and are representative of at least three independent experiments. <b>f</b>): Concentrated conditioned media normalised against cell numbers, analysed for LIF expression.</p

Analysis of morphology and migration of fibrosarcoma cells exposed to contractility inhibitors.

<p><b>a</b>): Cells were treated with P6 (5 µM), blebbistatin (5 µM) or Y27632 (10 µM) for 18 h. Representative images of P6 treated cells by phase contrast microscopy; blebbistatin-treated cells stained for F-actin; Y27632 treated cells stained with CellMask Orange. Scale bars, 50 µm. <b>b</b>): Migration was quantified by measuring wound closure. Images were collected at 12 h for the fswt and fs188 cells, at 18 h for the fs120 cells and at 24 h for the fs164 cells to correlate with 40–60% wound closure in untreated conditions. Contractility inhibitors were added at the same concentrations used in (<b>a</b>) 18 h before the start of the assay and replaced in fresh media at assay start. Each point represents data obtained from 3 independent experiments ± SEM, each conducted with 2–4 replicates. *p<0.05, ***p<0.001 values represent significance over untreated cultures (ANOVA followed by Tukey-Kramer post-test). <b>c</b>): Cells were incubated overnight with P6 (5 µM). Equal amounts of proteins (30 µg) were analysed for integrin β1 (β-Itg) and pMLC. A representative immunoblot is shown.</p

Population doubling times (hours) of fibrosarcoma cells grown on 2D surfaces.

<p>Population doubling times (hours) of cells grown on plastic, collagen or fibronectin are means of 3–6 independent experiments ± SEM.</p><p><b>*</b>p<0.05,</p><p>**p<0.01,</p><p>***p<0.001 values represent differences between fswt/fs188 cells versus fs164/fs120 cells (two way ANOVA followed by Bonferroni post-test).</p