The previous results indicated that in vitro the EGFR could not compensate for an acute reduction in IGF1R expression. We therefore sought to determine effects on EGFR activation following chronic IGF1R knockdown. MCF7, MDA-MB-231 and MDA-MB-468
TG-101348 were stably transfected with pSUPER constructs expressing IGF1R or control shRNAs. We were unable to induce a stable phenotype of long-term IGF1R knockdown in MDA-MB-231 and MDA-MB-468 cells (not shown), whereas stable MCF7 clones transfected with pSUPER expressing IGF1R shRNA exhibited an ～80% reduction in IGF1R levels compared to control transfectants. This knockdown was maintained for at least 8–12 weeks compared to transient knockdown which lasted for up to 7 days (not shown). The degree of stable knockdown was comparable to that observed 48 h after transient IGF1R knockdown using 200 nM IGF1R siRNA ( Fig. 4A, left). While transient IGF1R gene silencing reduced MCF7 cell survival to 40% of control levels, the effect of stable knockdown was greater (20% of control levels; Fig. 4A, right), likely reflecting the shorter duration of transient IGF1R knockdown and the clonal nature of the stable clones. Because MCF7
cells express low levels of endogenous EGFR, we transfected MCF7 control and IGF1R shRNA clones with an empty vector (EV) or HA-tagged full-length EGFR to investigate the ability of the EGFR to influence clonogenic survival following long-term IGF1R knockdown. Consistent with results of transient IGF1R knockdown, stable IGF1R knockdown in EV-transfected MCF7 cells resulted in significant inhibition of clonogenic survival. In cells overexpressing the EGFR we could detect increased phosphorylation of EGFR Y1068 ( Fig. 4B, left), but as in MDA-MB-468 cells this appeared not to induce an effective survival signal, since the survival of the IGF1R shRNA clones was similar following transfection with EV or EGFR ( Fig. 4B, right).
