We have previously demonstrated that neuroblastoma cells increase the expression of interleukin-6 by bone marrow stromal cells and that stimulation does not require cell-cell contact. In this study we report the purification and identification of a protein secreted by neuroblastoma cells that stimulates interleukin-6 production by stromal cells. Using a series of chromatographic purification steps including heparin-affinity, ion exchange, and molecular sieve chromatography followed by trypsin digestion and liquid chromatography tandem mass spectrometry, we identified in serum-free conditioned medium of neuroblastoma cells several secreted peptides including galectin-3-binding protein, also known as 90-kDa Mac-2-binding protein. We demonstrated the presence of the galectin-3-binding protein in the conditioned medium of several neuroblastoma cell lines and in chromatographic fractions with interleukin-6 stimulatory activity. Consistently, bone marrow stromal cells express galectin-3, the receptor for galectin-3-binding protein. Supporting a role for galectin-3-binding protein in stimulating interleukin-6 expression in bone marrow stromal cells, we observed that recombinant galectin-3-binding protein stimulated interleukin-6 expression in these cells and that interleukin-6 stimulation by neuroblastoma-conditioned medium was inhibited in the presence of lactose or a neutralizing anti-galectin-3 antibody. Down-regulation of galectin-3-binding protein expression in neuroblastoma cells also decreased the interleukin-6 stimulatory activity of the conditioned medium on bone marrow stromal cells. We also provide evidence that stimulation of interleukin-6 by galectin-3-binding protein involves activation of the Erk1/2 pathway. The data, thus, identifies galectin-3-binding protein as a factor secreted by neuroblastoma cells that stimulates the expression of interleukin-6 in bone marrow stromal cells and provides a novel function for this protein in cancer progression.It has become increasingly evident that the tumor microenvironment plays an important contributory role to cancer progression (1). Tumor cells closely interact with non-malignant cells present in the tumor stoma through a series of adhesiondependent and adhesion-independent mechanisms. Among the adhesion-independent interactions, cytokines, chemokines, and growth factors play a critical role (2, 3). In this regard, the bone marrow, which is a frequent site of metastasis, provides a microenvironment that is particularly favorable to cancer progression because it is a source of hematopoietic and mesenchymal cells that produce a large number of cytokines and chemokines (4 -6).Neuroblastoma is a neural crest-derived cancer that is the second most common solid tumor in children (7). At the time it is diagnosed, it has metastasized in 56% of the cases, primarily to the bone marrow, the bone, and the liver (8). The mechanisms by which neuroblastoma cells invade the bone have been recently elucidated by several laboratories, including our group (9 -11). We have previou...

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There is recent evidence that bone marrow-derived mesenchymal cells (BMMC) play an important role in bone metastasis in several cancers, including myeloma and neuroblastoma. We previously reported that in neuroblastoma these cells are a source of interleukin-6 (IL-6) that promotes the growth and survival of neuroblastoma cells and bone degradation. We have recently isolated and identified Galectin-3 binding protein (Gal-3BP) as a soluble factor produced by a neuroblastoma cell line as a factor upregulating the production of IL-6 by BMMC. Here we document the production of Gal-3BP in several neuroblastoma cell lines, in particular those without MYCN amplification that correlates with their ability to induce the production of IL-6 in BMMC. The stimulation of IL-6 by Gal-3BP is mediated by Gal-3, a multifunctional glycoprotein that binds Gal-3BP and is present in BMMC. It is also dependent on the activation of the Ras/Raf/MEK/ERK1/2 pathway by Gal-3BP/Gal-3 and is blocked by the ERK1/2 inhibitor PD980559. Transcriptional activation of IL-6 by Gal-3BP/Gal-3 requires a 115 bp region (−97 to −212) in the IL-6 promoter that contains recognition motifs for a cAMP responsive element binding protein (CREB) and a CCAAT/enhancer binding protein (C/EBP). The expression of Gal-3BP was also examined by immunohistochemistry in 38 primary neuroblastoma tumors with (n=9) and without (n=29) MYCN amplification. This analysis revealed that Gal-3BP is paradoxically more abundant in MYCN non-amplified tumors. This unanticipated observation suggests a significant difference in the Gal-3BP/Gal-3/IL-6 pathway between subgroups of neuroblastoma tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 557.