Abstract A33: in ovarian cancer cell lines resistant to microtubule-stabilizing agents, expression of IGF2 and ABCB1 is increased and IGF1R inhibition restores drug sensitivity

Abstract

Abstract Overexpression of insulin-like growth factor 2 (IGF2) is associated with worse outcome in ovarian cancer patients; previously, our group has shown that IGF2 is upregulated after Taxol treatment of ovarian cancer cells and leads to activation of AKT. Thus, we hypothesized that IGF2 may be involved in drug resistance. The Taxol-resistant cell line HEY-T30 was developed in our laboratory from chemo-naive, sensitive HEY ovarian cancer cells by repeated Taxol treatment in tissue culture. HEY-T30 is 40-fold resistant to Taxol (IC50: 2.6 nM versus 105.9 nM), as determined by sulforhodamine B cytotoxicity assays. In these cells, IGF2 mRNA levels, measured by quantitative PCR, are increased 8.8-fold compared with its expression in the parental cell line. In addition, HEY-T30 have a genomic amplification of ABCB1 (mdr1; encoding the drug efflux pump p-glycoprotein) detected by array CGH, associated with increased ABCB1 mRNA expression. Alterations in IGF2 and ABCB1 were assessed in an additional drug-resistant ovarian cancer cell line, OVCAR8-D30, developed in our laboratory. OVCAR8-D30 is 2-fold-resistant to discodermolide (IC50: 56 nM versus 115 nM), another microtubule-stabilizing drug with a distinct chemical structure. This resistant cell line also showed an increase (4-fold) in IGF2 expression compared to its parental cell line, as well as a 19-fold increase in ABCB1 expression. To determine the effect of IGF pathway inhibition in Taxol-resistant cells, NVP-AEW541 (a small molecule inhibitor of the IGF1R, the major receptor for IGF2) was used. By concurrent treatment with NVP-AEW541, HEY-T30 cells were significantly re-sensitized to Taxol (IC50 from 105.9 nM to 17.0 nM). The effect of combined Taxol and NVP-AEW541 treatment on cell cycle distribution was evaluated by flow cytometry. Eighteen hours after Taxol (75 nM, 125 nM and 250 nM) and NVP-AEW541 (1 μM) treatment alone or in combination, more cells were arrested in G2/M with combination treatment compared to Taxol treatment alone at the higher Taxol concentrations (75 nM: 23.3% to 26.4% with NVP, 125 nM: 23.8% to 44.0% with NVP, and 250 nM: 34.6% to 74.1% with NVP) as determined by flow cytometry. At this time point, caspase activity analysis by flow cytometry detection of FAM-VAD-FMK FLICA reagent, showed no significant differences between the two treatments (75 nM: 6.7% and 9.5% with NVP, 125 nM: 10.0% and 11.5% with NVP, and 250 nM: 12.4% and 14.9% with NVP). These data suggest that treatment with the IGF1R inhibitor NVP-AEW541 can restore Taxol sensitivity in cells with multiple aberrations that confer drug-resistance (IGF2 and ABCB1 over-expression). We conclude that IGF2 has a prominent role in resistance to microtubule-stabilizing drugs in ovarian cancer cell lines and we therefore propose IGF2 to be a valuable target in future molecular therapies for chemo-resistant ovarian cancer. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A33