2247

FGF2

BFGF|FGFB|HBGF-2;fibroblast growth factor 2 (basic);FGF2;fibroblast growth factor 2 (basic)

basic fibroblast growth factor bFGF|heparin-binding growth factor 2|prostatropin

4q26

protein-coding

Overexpression of genes, through genomic amplification and other mechanisms, cancritically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that areprimarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was definedas containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in ALL cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primarysamples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and alteredthe cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4in Xenopus blastocysts was also enhanced. Our data are consistent with a role ofGPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads tomesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeuticintervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene.

We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were culturedwith FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions(E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events (increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, andsuppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer.