title: IGFBP-2 enhances VEGF gene promoter activity and consequent promotion of angiogenesis by neuroblastoma cells.
abstract: IGF binding protein (IGFBP)-2 is one of the most significant genes in the signature of major aggressive cancers. Previously, we have shown that IGFBP-2 enhances proliferation and invasion of neuroblastoma cells, suggesting that IGFBP-2 activates a protumorigenic gene expression program in these cells. Gene expression profiling in human neuroblastoma SK-N-SHEP (SHEP)-BP-2 cells indicated that IGFBP-2 overexpression activated a gene expression program consistent with enhancement of tumorigenesis. Regulation was significant for genes involved in proliferation/survival, migration/adhesion, and angiogenesis, including the up-regulation of vascular endothelial growth factor (VEGF) mRNA (>2-fold). Specific transcriptional activation of the VEGF gene by IGFBP-2 overexpression was demonstrated via cotransfection of a VEGF promoter Luciferase construct in SHEP-BP-2. Cotransfection of VEGF promoter Luciferase construct with IGFBP-2 protein in wild-type SHEP cells indicated that transactivation of VEGF promoter only occurs in the presence of intracellular IGFBP-2. Cell fractionation and immunofluorescence in SHEP-BP-2 cells demonstrated nuclear localization of IGFBP-2. These findings suggest that transcriptional activation of VEGF promoter is likely to be mediated by nuclear IGFBP-2. The levels of secreted VEGF (up to 400 pg/10(6) cells) suggested that VEGF might elicit angiogenic activity. Hence, SHEP-BP-2 cells and control clones cultured in collagen sponge were xenografted onto chick embryo chorioallantoic membrane. Neomicrovascularization was observed by 72 h, solely in the SHEP-BP-2 cell xenografts. In conclusion, our data indicate that IGFBP-2 is an activator of aggressive behavior in cancer cells, involving nuclear entry and activation of a protumorigenic gene expression program, including transcriptional regulation of the VEGF gene and consequent proangiogenic activity of NB cell xenografts in vivo.
authors.affiliation: Centre for Hormone Research, Cell Biology, Murdoch Childrens Research Institute, Royal Children's Hospital, Flemington Road, Parkville 3052, Victoria, Australia.
authors.forename: Walid J
authors.initials: WJ
authors.lastname: Azar
authors.forename: Sheena H X
authors.initials: SH
authors.lastname: Azar
authors.forename: Sandra
authors.initials: S
authors.lastname: Higgins
authors.forename: Ji-Fan
authors.initials: JF
authors.lastname: Hu
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Hoffman
authors.forename: Donald F
authors.initials: DF
authors.lastname: Newgreen
authors.forename: George A
authors.initials: GA
authors.lastname: Werther
authors.forename: Vincenzo C
authors.initials: VC
authors.lastname: Russo
mesh_terms: D002458:Cell Fractionation; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000235:genetics* / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics* / Q000378:metabolism
chemical_list: D018971:Insulin-Like Growth Factor Binding Protein 2; D042461:Vascular Endothelial Growth Factor A