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.
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.
Higgins
S
S
Centre for Hormone Research, Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville 3052, Victoria, Australia.
Wong
S H X
SH
Richner
M
M
Rowe
C L
CL
Newgreen
D F
DF
Werther
G A
GA
Russo
V C
VC
D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000502:physiology*; D005109:Extracellular Matrix / Q000502:physiology; D016222:Fibroblast Growth Factor 2 / Q000502:physiology*; D016193:G1 Phase / Q000187:drug effects*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051798:Inhibitor of Differentiation Protein 1 / Q000502:physiology*; D051797:Inhibitor of Differentiation Protein 2 / Q000502:physiology*; D051796:Inhibitor of Differentiation Proteins / Q000502:physiology*; D009363:Neoplasm Proteins / Q000502:physiology*; D009447:Neuroblastoma / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction
D051792:Basic Helix-Loop-Helix Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016222:Fibroblast Growth Factor 2; D051798:Inhibitor of Differentiation Protein 1; D051797:Inhibitor of Differentiation Protein 2; D051796:Inhibitor of Differentiation Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; C497453:ID1 protein, human; C497450:ID2 protein, human; C490553:NEUROD6 protein, human; C075640:ID3 protein, human