IGF2 is required for YAP-mediated G2/M arrest override and cell survival and proliferation after irradiation
To address whether IGF2/Akt activity regulates the YAP-associated DNA damage response defect after radiation, we used Shh-treated CGNP cultures. Confirming our observations (Figure 5), YAP infection is associated with increased Akt activity (Figure 6A) as determined by phosphorylation of S473. Treatment of YAP-infected CGNPs with the drug LY294002, which inhibits phosphoinositide-3 kinase, the upstream activator of Akt, reduced levels of S473-phosphorylated Akt. After irradiation, we saw induction of ATM phosphorylation in GFP- and YAP-infected CGNPs, although to a lesser extent in the presence of ectopic YAP, indicating reduced activity of this kinase. In keeping with reduced ATM activity in the presence of YAP, we also observed reduced Chk2 phosphorylation. In the presence of LY294002, full ATM and Chk2 phosphorylation were recovered, indicating that YAP requires Akt activity for its suppressive effect on ATM and Chk2.
We next wished to determine whether IGF2 is necessary for the effects of YAP on the DNA damage response. To this end, we used retroviruses targeting IGF2 for short hairpinRNA-mediated knock-down. As shown in Figure 6B, YAP-infected CGNPs transduced with these retroviruses showed strikingly reduced levels of IGF2, in comparison with YAP-infected CGNPs transduced with retroviruses carrying a scrambled, non-specific short hairpin RNA sequence. Consistent with IGF2 being an upstream activator of Akt, IGF2 knockdown was associated with reduced Akt Ser473 phosphorylation. In addition, knocking down IGF2 in YAP-expressing cells led to a recovery of phospho-ATM and phospho-Chk2 levels, as well as phospho-Cdk1, comparable to those observed in GFP-infected cells. The increase in inactive Cdk1 when IGF2 is knocked down indicates cells arresting after radiation, due to a restored G2/M checkpoint. IGF2 knock-down also blocked the effects of YAP on DNA damage-dependent focus formation after irradiation (Figure 6C), causing a significant rescue of focus formation as determined by immunofluorescent staining for 53BP1. Moreover, IGF2 knock-down impaired the effects of YAP expression on CGNP survival after irradiation, as well as proliferation (Figure 6D). These results are consistent with IGF2 and its downstream effector Akt being necessary for the ability of YAP to inactivate the G2/M checkpoint, permitting ongoing proliferation and enhancing survival of irradiated cells.