Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth
Erik H. Knelson, … , Stephen G. Marcus, Gerard C. Blobe
Erik H. Knelson, … , Stephen G. Marcus, Gerard C. Blobe
Published June 17, 2014
Citation Information: J Clin Invest. 2014;124(7):3016-3031. https://doi.org/10.1172/JCI74270.
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Research Article Oncology

Stromal heparan sulfate differentiates neuroblasts to suppress neuroblastoma growth

Abstract

Neuroblastoma prognosis is dependent on both the differentiation state and stromal content of the tumor. Neuroblastoma tumor stroma is thought to suppress neuroblast growth via release of soluble differentiating factors. Here, we identified critical growth-limiting components of the differentiating stroma secretome and designed a potential therapeutic strategy based on their central mechanism of action. We demonstrated that expression of heparan sulfate proteoglycans (HSPGs), including TβRIII, GPC1, GPC3, SDC3, and SDC4, is low in neuroblasts and high in the Schwannian stroma. Evaluation of neuroblastoma patient microarray data revealed an association between TGFBR3, GPC1, and SDC3 expression and improved prognosis. Treatment of neuroblastoma cell lines with soluble HSPGs promoted neuroblast differentiation via FGFR1 and ERK phosphorylation, leading to upregulation of the transcription factor inhibitor of DNA binding 1 (ID1). HSPGs also enhanced FGF2-dependent differentiation, and the anticoagulant heparin had a similar effect, leading to decreased neuroblast proliferation. Dissection of individual sulfation sites identified 2-O, 3-O-desulfated heparin (ODSH) as a differentiating agent, and treatment of orthotopic xenograft models with ODSH suppressed tumor growth and metastasis without anticoagulation. These studies support heparan sulfate signaling intermediates as prognostic and therapeutic neuroblastoma biomarkers and demonstrate that tumor stroma biology can inform the design of targeted molecular therapeutics.

Authors

Erik H. Knelson, Angela L. Gaviglio, Jasmine C. Nee, Mark D. Starr, Andrew B. Nixon, Stephen G. Marcus, Gerard C. Blobe

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Figure 6

Heparin promotes neuroblast differentiation via FGFR1, ERK, and ID1.

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Heparin promotes neuroblast differentiation via FGFR1, ERK, and ID1. (A)...
(A) Phase-contrast images of 5Y and BE2 cells after 72 hours of heparin treatment (1 μg/ml). Arrows identify abnormally long neurites (>2× the mean neurite length). Original magnification, ×10; scale bar: 100 μm. Quantification of neurite length using NeuronJ software. Data are presented as the mean of 3 fields ± SEM. **P < 0.01, ***P < 0.001 versus control, by 2-tailed Student’s t test. (B) Western blots for differentiation markers in 5Y and SK-N-AS treated for 72 hours with a dose course of heparin or ATRA (10 μM). Densitometry for NF160 normalized to β-actin is shown as the percentage of control. (C) Western blots for differentiation markers and ID1 in 5Y and SK-N-AS treated for 72 hours with heparin (1 μg/ml), FGF2 (1 ng/ml), PD173074, UO126, CI1040, or SU5402 (1 μM). Transient transfection with dominant negative FGFR1 or IRES-GFP control. GFP fluorescence was used to confirm construct expression. Densitometry for NF160 normalized to β-actin is shown as the percentage of control.