Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation, migration, and invasion.

IGF binding proteins (IGFBPs) modulate IGF cellular bioavailability and may directly regulate tumor growth and invasion. We have previously shown that IGFBP-2 binds and localizes IGF-I to the pericellular matrix and have provided some evidence suggesting that the heparin binding domain (HBD) or the arginine-glycine-aspartic acid (RGD) integrin binding motif may be involved in these interactions. However, the precise mechanisms involved remain to be elucidated. We therefore mutated the HBD or RGD sequence of IGFBP-2 and investigated consequent effects on extracellular matrix (ECM) binding, IGF-induced proliferation, and migration of neuroblastoma cells. IGFBP-2 and its arginine-glycine-glutamic acid (RGE) mutant similarly bound ECM components, whereas binding of mutant HBD-IGFBP-2 to each of the ECM substrates was markedly reduced by 70-80% (P < 0.05). IGF-I (100 ng/ml) increased incorporation of 3H-thymidine in neuroblastoma SK-N-SHEP cells by approximately 30%, an effect blunted by exogenously added native or either mutant IGFBP-2. Overexpression of IGFBP-2 and its RGE mutant potently promoted SHEP cell proliferation (5-fold), whereas SHEP cell proliferation was negligible when HBD-IGFBP-2 was overexpressed. Addition or overexpression of IGFBP-2 and its RGE mutant potently (P < 0.05) enhanced SHEP cell migration/invasion through the ECM. However, overexpression of the HBD-IGFBP-2 mutant potently inhibited (50-60%) SHEP cell invasion through ECM. Thus, IGFBP-2, which binds to the ECM, enhances proliferation and metastatic behavior of neuroblastoma cells, functions that directly or indirectly use the HBD but not the integrin binding sequence. Our novel findings thus point to a key role for the HBD of IGFBP-2 in the control and regulation of neuroblastoma growth and invasion.

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells.

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.