Fibroblast growth factor 2 reactivates G1 checkpoint in SK-N-MC cells via regulation of p21, inhibitor of differentiation genes (Id1-3), and epithelium-mesenchyme transition-like events

Endocrinology. 2009 Sep;150(9):4044-55. doi: 10.1210/en.2008-1797. Epub 2009 May 28.

Abstract

We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions (E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events (increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, and suppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Cell Differentiation / genetics*
  • Cell Line, Tumor
  • Cyclin-Dependent Kinase Inhibitor p21 / physiology*
  • Extracellular Matrix / physiology
  • Fibroblast Growth Factor 2 / physiology*
  • G1 Phase / drug effects*
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Inhibitor of Differentiation Protein 1 / physiology*
  • Inhibitor of Differentiation Protein 2 / physiology*
  • Inhibitor of Differentiation Proteins / physiology*
  • Neoplasm Proteins / physiology*
  • Neuroblastoma / genetics
  • RNA Interference
  • RNA, Messenger / metabolism
  • Signal Transduction

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • Cyclin-Dependent Kinase Inhibitor p21
  • ID1 protein, human
  • ID2 protein, human
  • Inhibitor of Differentiation Protein 1
  • Inhibitor of Differentiation Protein 2
  • Inhibitor of Differentiation Proteins
  • NEUROD6 protein, human
  • Neoplasm Proteins
  • RNA, Messenger
  • Fibroblast Growth Factor 2
  • ID3 protein, human