Abstract #1744: Inhibition of the insulin-like growth factor 1 receptor (IGF1R) induces cell cycle arrest and apoptosis in Wilms tumor cells

Background: We have identified DNA copy number gain and overexpression of the insulin-like growth factor 1 receptor (IGF1R) to be significantly correlated with Wilms tumour relapse. Coupled with autocrine/paracrine activation by the ligand IGF2, frequently overexpressed in Wilms tumours by genetic and epigenetic means, this receptor tyrosine kinase represents an excellent novel therapeutic target. In this study we investigated the growth inhibitory effects and mechanisms of action of two recently developed specific IGF1R inhibitors, NVP-AEW541 and picropodophyllin (PPP), on Wilms tumour cells in vitro. Methods: The cell lines used included human embryonic kidney (HEK293), Wilms tumour (WiT49, STA2A) and mouse embryonic fibroblasts R- (IGF1R negative) and R+ (IGF1R positive). Levels of phosphorylated receptor tyrosine kinases were assayed using the Phospho-RTK Array (R&D Systems). Cell viability evaluated by the MTS assay. Downstream signalling components were assessed by Western blotting and electrochemiluminescence (MSD). Cell cycle analysis was carried out by FACS, and apoptosis assessed by evaluation of caspase 3 cleavage. Results: We observed high levels of expression and constitutive activation of IGF1R and IRS1 in HEK293, WiT49, STA2A and R+ cells relative to the negative control R- line. Both PPP and NVP-AEW541 showed an explicit inhibitory effect towards these cell lines, with IC50 values of 0.3-0.4µM and 0.6-1.0µM respectively. R- cells were insensitive to both compounds. In serum-starved cells, IGF2 stimulation increased cell viability and activated downstream pathways via increased levels of phosphorylated IGF1R, IRS1, Akt and Erk1/2, effects which were abrogated by both PPP and NVP-AEW541 at 1x and 3x IC50. Targeting the receptor with specific siRNAs decreased cell viability, diminished signalling via Akt and MAPK pathways, induced cell cycle arrest at the G1/S checkpoint, and resulted in apoptosis. These effects were replicated by NVP-AEW541, however PPP instead induced a profound arrest in the G2/M phase. Conclusions: Selective inhibition of IGF1R represents a potentially useful therapeutic strategy in relapsed Wilms tumours. Compounds which target the receptor down-regulate key downstream signalling pathways, induce cell cycle arrest and lead to apoptosis, however appear to have different mechanisms of action.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1744.