Balance of pro- versus anti-angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth

Abstract

Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro. VEGF(165) has been shown to be the predominant angiogenic isoform expressed in NB cell lines and tumours. In this study, we investigated the anti-angiogenic isoform of VEGF-A, generated from distal splice site selection in the terminal exon of VEGF (VEGF(165)b) and shown to be down-regulated in epithelial malignancies. The expression of both the pro- (VEGF(xxx)) and the anti-angiogenic (VEGF(xxx)b) isoforms was compared in a range of NB and ganglioneuroma (GN) tumours. Whereas VEGF(xxx)b and VEGF(xxx) were both expressed in GN, specific up-regulation of the VEGF(xxx) isoforms was seen in NB at RNA and protein levels. Highly tumourigenic NB cell lines also showed up-regulation of the angiogenic isoforms relative to VEGF(xxx)b compared to less tumourigenic cell lines, and the isoforms were differentially secreted. These results indicate that VEGF(165) is up-regulated in NB and that there is a difference in the balance of isoform expression from anti-angiogenic VEGF(165)b to angiogenic VEGF(165). Treatment with recombinant human VEGF(165)b significantly reduced the growth rate of established xenografts of SK-N-BE(2)-C cells (4.24 +/- 1.01 fold increase in volume) compared with those treated with saline (9.76 +/- 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF(165)b-treated tumours (19.4 +/- 1.9 vessels/mm(3)) in contrast to the saline-treated tumours (45.5 +/- 8.6 vessels/mm(3)). VEGF(165)b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK-N-BE(2)-C cells after 48 h. In conclusion, VEGF(165)b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of VEGF(165)b in NB and other paediatric malignancies.

Figure 1. Organisation of the VEGF-A gene

A. Gene structure. TSS=transcriptional start site. B. mRNA species. Alternative splicing of VEGF-A gene in the terminal exon results in two families of isoforms, the pro-angiogenic VEGF_xxx, and the anti-angiogenic VEGF_xxxb isoforms. AUG= start site for translation, UTR=untranslated region, pA = poly adenylation site. C. Protein structure of two major isoforms of each family. This C-terminal splicing leads to an alternative last six amino acids (CDKPRR or SLTRKD). The isoforms are termed according to the amino acid number of the resulting protein (xxx). HSPG=heparin sulphate proteoglycan, R1=VEGF receptor 1, R2 =VEGF Receptor 2 (not to scale).

Figure 2. VEGF_xxx is up-regulated in neuroblastoma but not ganglioneuroma at mRNA and protein levels

(A) Extraction of mRNA from neuroblastic tumour samples followed by RT-PCR using primers to detect both families of isoforms. (B) Relative amounts of VEGF_xxx mRNA in neuroblastic tumour samples, NB and GN, represented as mean ± SEM. qPCR values were calculated in relation to expression of three reference genes (SDHA, HPRT I and β-actin). VEGF_xxx was 3.54 times greater in malignant tumours than in benign tumours. (C) ELISA results on total VEGF and VEGF_xxxb protein expression by different neuroblastic tumour samples showed as data points and median. Results normalized by protein. (D) VEGF_xxxb relative expression decreases significantly with the malignancy of the tumour (unpaired t test, p<0.05). Mean ± SEM. (E) Immunohistochemical staining of VEGF_xxxb and total VEGF in neuroblastic tumours. (F) Relative amounts of SRPK1 mRNA in NB and GN, represented as mean ± SEM. qPCR values were calculated in relation to expression of three reference genes (SDHA, HPRT I and β-actin). SRPK1 was 4.58 times greater in NB than in GN.

Figure 3. Differential expression and secretion of VEGF isoforms by neuroblastoma cell lines

(A) Extraction of mRNA from NB cell lines followed by RT-PCR using primers to detect both families of isoforms. FAG is fetal adrenal gland and LEC, lymphatic endothelial cells. (B) ELISA for VEGF_xxxb and total VEGF on cell lysates for different NB cell lines where MYCN oncogene amplification is associated with poor prognosis. Results normalized to protein. HEK293 cells were used as positive control for the expression of VEGF xxxb. (C) VEGF_xxxb relative expression decreases significantly in highly tumorigenic, MYCN amplified cell lines (One-way ANOVA, post test for linear trend, p<0,01). Results normalized to HEK293 cells. (D) Cell ELISA for VEGF_xxxb and total VEGF showing a significant secretion of VEGF (1-20fg/cell) in all neuroblastoma cell lines. VEGF_xxxb secretion was 1-2 orders of magnitude lower (0.2-0.6fg/cell). Results normalized by cell number. (E) Proportion of secreted VEGF that is VEGF_xxxb. Only SH-IN and SH-EP cells secreted more than 5% of its VEGF as VEGF_xxxb.

Figure 4. rhVEGF₁₆₅b reduces tumour growth rate when administered subcutaneously twice a week

(A) Treatment of established murine NB tumours with either saline or rhVEGF₁₆₅b (100μg subcutaneously twice a week). Values of tumour fold increase in volume were reduced in the VEGF₁₆₅b-treated group in comparison with saline-treated controls (from 9.76±3.58 to 4.24±1.01 on day 14, ANOVA, p<0.001). (B) VEGFR2 immunohistochemical staining of mice tumours. The staining was mostly detected in the vascular endothelium (in brown). (C, D) Quantification of the blood vessel number and blood vessel area present in the saline-treated tumours versus the VEGF₁₆₅b-treated tumours. Treatment with rhVEGF₁₆₅b decreased the vessel density and the area of the blood vessels in the tumours (unpaired t test, p<0,05).

Figure 5. rhVEGF₁₆₅b has no effect on SK-N-BE(2)C neuroblastoma cells

Results normalized by serum-free media control. Data represent mean ± SEM. After 24 hours or 48 hours, SK-N-BE(2)C cells treated with 0.5, 1, 2.5 and 5 nM did not show any significant difference at (A) viability, (B) proliferation, (C) apoptosis or (D) cytotoxicity levels (Student’s t test, p>0.05). Representative data for one concentration of rhVEGF₁₆₅b (1nM) and one time point is shown.