FGF-2 RE-ACTIVATES G1-CHECKPOINT IN SK-N-MC CELLS VIA REGULATION OF p21, INHIBITOR OF DIFFERENTIATION GENES (Id1-3), AND EMT-LIKE EVENTS.
Endocrinology. 2009 May 28;
Authors: Higgins S, Wong SH, Richner M, Rowe CL, Newgreen DF, Werther GA, Russo VC
We have recently demonstrated that FGF-2 promotes neuroblastoma cell differentiation and over-rides their mitogenic response to IGF-I. However the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50ng/ml) and/or IGF-I (100ng/ml) up to 48h. FACS 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 EMT interconversion. 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 EMT-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.
PMID: 19477940 [PubMed - as supplied by publisher]