Type III TGF-β receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma
Erik H. Knelson, Angela L. Gaviglio, Alok K. Tewari, Michael B. Armstrong, Karthikeyan Mythreye, Gerard C. Blobe
Erik H. Knelson, Angela L. Gaviglio, Alok K. Tewari, Michael B. Armstrong, Karthikeyan Mythreye, Gerard C. Blobe
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Research Article Oncology

Type III TGF-β receptor promotes FGF2-mediated neuronal differentiation in neuroblastoma

Abstract

Growth factors and their receptors coordinate neuronal differentiation during development, yet their roles in the pediatric tumor neuroblastoma remain unclear. Comparison of mRNA from benign neuroblastic tumors and neuroblastomas revealed that expression of the type III TGF-β receptor (TGFBR3) decreases with advancing stage of neuroblastoma and this loss correlates with a poorer prognosis. Patients with MYCN oncogene amplification and low TGFBR3 expression were more likely to have an adverse outcome. In vitro, TβRIII expression was epigenetically suppressed by MYCN-mediated recruitment of histone deacetylases to regions of the TGFBR3 promoter. TβRIII bound FGF2 and exogenous FGFR1, which promoted neuronal differentiation of neuroblastoma cells. TβRIII and FGF2 cooperated to induce expression of the transcription factor inhibitor of DNA binding 1 via Erk MAPK. TβRIII-mediated neuronal differentiation suppressed cell proliferation in vitro as well as tumor growth and metastasis in vivo. These studies characterize a coreceptor function for TβRIII in FGF2-mediated neuronal differentiation, while identifying potential therapeutic targets and clinical biomarkers for neuroblastoma.

Authors

Erik H. Knelson, Angela L. Gaviglio, Alok K. Tewari, Michael B. Armstrong, Karthikeyan Mythreye, Gerard C. Blobe

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Figure 4

TβRIII promotes neuronal differentiation via FGF2 signaling.

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TβRIII promotes neuronal differentiation via FGF2 signaling. (A) Western...
(A) Western blots for differentiation markers and graph of neurite analysis using NeuronJ (mean ± SEM) in 5Y cells expressing nontargeted shRNA or shRNA against TβRIII for 96 hours, with or without 10 ng/ml FGF2 treatment (gray bars). Densitometry for NF160 normalized to β-actin is shown as percent control. P < 0.001 for main effect receptor (2-way ANOVA); P < 0.01 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (B) Western blot for differentiation markers in 5Y cells following 96 hours of adenoviral transduction with GFP control, TβRIII-GFP, TβRIII-HA, or mutant TβRIII-HA lacking GAG chain attachment sites (TβRIII-ΔGAG). Differentiation markers in SHEP cells following 72-hour TβRIII knockdown and rescue. Densitometry for NF160 normalized to β-actin is shown as percent control. NTC, nontargeted control. (C) Western blots in 5Y cells for differentiation markers and phosphorylated and total Erk following 96 hours of transduction and treatment with 1 ng/ml FGF2 (gray bars). Quantification of 3 independent experiments (mean ± SEM). P < 0.0001 for main effect receptor (2-way ANOVA); P < 0.001 for main effect FGF2 (2-way ANOVA); interaction P < 0.05. (D) I125 FGF2 binding and crosslinking with total cell lysate (TCL) and TβRIII pull-down (TβRIII IP) in 5Y and SHEP cell lines. Arrows for total cell lysate mark FGFR1 (145 kDa) and TβRIII (80 kDa). Dose course of I125 FGF2 (1 ng/ml, 5 ng/ml, 10 ng/ml); dose course of cold FGF2 (50 ng/ml, 100 ng/ml, 500 ng/ml); GFP condition treated with 10 ng/ml I125 FGF2. Densitometry for TβRIII normalized to β-actin is shown as percent control. (E) Coimmunoprecipitation of TβRIII-HA and FGFR1-FLAG in SHEP cells. PAS beads were used as control.