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 3

TβRIII promotes neuronal differentiation of NB cells.

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TβRIII promotes neuronal differentiation of NB cells. Transient transduc...
Transient transductions with TβRIII-GFP, GFP control, nontargeted control shRNA (shNTC), or shRNA to TβRIII (shTβRIII). (A) Phase microscopy of 5Y cells 96 hours after plating. Original magnification, ×10; scale bar: 100 μM. (B) Time course of 5Y cell neurite length (mean of 3 fields ± SEM). Adenoviral transduction at 24 hours. P < 0.0001 for main effects of time and receptor expression (2-way ANOVA); interaction P < 0.05; *P < 0.05, **P < 0.01, ***P < 0.001 (Bonferroni post-hoc comparisons shown for TβRIII-GFP compared to GFP and control). (C) 5Y cell neurite length (mean of 3 fields ± SEM) after 96 hours of TβRIII knockdown. ****P < 0.0001 (2-tailed Student’s t test). (D) Western blot for neurofilament 160 kDa (NF160), tyrosine hydroxylase (TH), neuron-specific enolase (NSE), β3-tubulin, and GAP43 after 96-hour transduction. Densitometry for NF160 normalized to β-actin is shown as percent control. (E) Quantification of differentiation markers from 3 independent experiments in 5Y cells normalized to β-actin (mean increase above control ± SEM). P < 0.05 for all markers (1-sample Student’s t test). (F) Differentiation markers after 72-hour TβRIII knockdown and rescue with knockdown-resistant rat TβRIII (rTβRIII). Densitometry for NF160 normalized to β-actin is shown as percent control. (G) Quantification of NF160 from 3 independent experiments (mean ± SEM) in SHEP cells normalized to β-actin. *P < 0.05 (1-sample t test and 2-tailed Student’s t test). (H) Microarray data set expression of SOX10 in tumors with low (bottom 10%) and high (top 10%) TGFBR3 expression (median [horizontal bars] and interquartile range [boxes]). ***P < 0.001 (Mann-Whitney).