Neuroblastoma progression correlates with downregulation of the lymphangiogenesis inhibitor sVEGFR-2

Abstract

Purpose: Tumor progression correlates with the induction of a dense supply of blood vessels and the formation of peritumoral lymphatics. Hemangiogenesis and lymphangiogenesis are potently regulated by members of the vascular endothelial growth factor (VEGF) family. Previous studies have indicated the upregulation of VEGF-A and -C in progressed neuroblastoma, however, quantification was performed using semiquantitative methods, or patients who had received radiotherapy or chemotherapy were studied.

Experimental design: We have analyzed primary neuroblastoma from 49 patients using real-time reverse transcription-PCR and quantified VEGF-A, -C, and -D and VEGF receptors (VEGFR)-1, 2, 3, as well as the soluble form of VEGFR2 (sVEGFR-2), which has recently been characterized as an endogenous inhibitor of lymphangiogenesis. None of the patients had received radiotherapy or chemotherapy before tumor resection.

Results: We did not observe upregulation of VEGF-A, -C, and -D in metastatic neuroblastoma, but found significant downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic stages III, IV, and IVs. In stage IV neuroblastoma, there were tendencies for the upregulation of VEGF-A and -D and the downregulation of the hemangiogenesis/lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2 in MYCN-amplified tumors. Similarly, MYCN transfection of the neuroblastoma cell line SH-EP induced the upregulation of VEGF-A and -D and the switching-off of sVEGFR-2.

Conclusion: We provide evidence for the downregulation of the lymphangiogenesis inhibitor sVEGFR-2 in metastatic neuroblastoma stages, which may promote lymphogenic metastases. Downregulation of hemangiogenesis and lymphangiogenesis inhibitors VEGFR-1 and sVEGFR-2, and upregulation of angiogenic activators VEGF-A and VEGF-D in MYCN-amplified stage IV neuroblastoma supports the crucial effect of this oncogene on neuroblastoma progression.

Fig. 1

Expression of VEGF ligands and receptors in 49 primary NB specimens as measured by real-time RT-PCR. A) VEGF-A, B) VEGF-C, C) VEGF-D, D) VEGFR-1, E) VEGFR-2, F) VEGFR-3, G) sVEGFR-2. Note statistically significant down-regulation of sVEGFR-2 in metastatic stages 3, 4 and 4s. None of the other molecules exhibits any obvious stage-specific regulation. Relative expression ± standard error are shown.

Fig. 2

Comparison of the expression of VEGF ligands and receptors in stage 4 NB with normal vs. amplified MYCN expression. A) VEGF-A, B) VEGF-C, C) VEGF-D, D) VEGFR-1, E) VEGFR-2, F) VEGFR-3, G) sVEGFR-2. There are no statistically significant differences, but tendencies for the up-regulation of VEGF-A and VEGF-D, and the down-regulation of VEGFR-1 and sVEGFR-2 in MYCN-amplified tumors. Relative expression ± standard error are shown.

Fig. 3

Expression of VEGF ligands and receptors in 24 NB cell lines as measured by real-time RT-PCR. A) VEGF-A, B) VEGF-C, C) VEGF-D, D) VEGFR-1, E) VEGFR-2, F) sVEGFR-2. Data for VEGFR-3 are not shown, because this receptor was almost undetectable. Expression of CHLA20 cells was used as reference and set as 1. Note that WAC2 are stably MYCN-transfected SH-EP cells. There is up-regulation of VEGF-A and VEGF-D, and a switch-off of sVEGFR-2 in WAC2.

Fig. 4

Comparison of the expression of VEGF ligands and receptors in 24 NB cell lines, sorted by their MYCN copy numbers (normal vs. amplified). A) VEGF-A, B) VEGF-C, C) VEGF-D, D) VEGFR-1, E) VEGFR-2, F) sVEGFR-2. There are no statistically significant differences, but tendencies for the down-regulation of VEGF-A, VEGF-C and VEGFR-2 in MYCN-amplified cell lines. However, this does not reflect our observations on primary NBs. Relative expression ± standard error are shown.