DNA-PKcs is activated under nutrient starvation and activates Akt, MST1, FoxO3a, and NDR1.

ABSTRACT

BACKGROUND: Presence of unperfused regions containing cells under hypoxia and nutrient starvation; contributes to radioresistance in solid human tumors. We have previously reported that cultured cells; under nutrient starvation show resistance to ionizing radiation compare with cells under normal; condition, and that nutrient starvation increases ATM activity, which causes cellular resistance to; ionizing radiation (Murata et al., BBRC2018). For further investigation of molecular mechanisms; underlying radioresistance of cells under nutrient starvation, effects of nutrient starvation on activity; of DNA-PKcs have been investigated because both DNA-PKcs and ATM belong to the PIKK family; and are required for DNA DSBs repair. In addition to DNA-PKcs, effects of nutrient starvation on; activities of FoxO3a and its regulators Akt, MST1 and AMPK have been investigated because FoxO3a; mediates cellular responses to stress and is activated under nutrient starvation. METHODS: A human glioblastoma cell line, T98G was used to examine the effects of nutrient starvation on activities and expression of DNA-PKcs, Akt, MST1, FoxO3a, NDR1, and AMPK. To elucidate; signal transduction pathways for FoxO3a activation under nutrient starvation, we examined effects of; specific inhibitors or siRNA for DNA-PKcs or Akt on activities and expression of MST1, FoxO3, NDR1, andAMPK. RESULTS: Under nutrient starvation, phosphorylations of DNA-PKcs at Ser2056, Akt at Ser473, MST at Thr183, FoxO3a at Ser413, NDR1 at Ser281 and Thr282, and AMPK at Thr172 were increased, which suggests their activation. Nutrient starvation did not affect expression of DNA-PKcs, Akt, MST1, or NDR1, with decreased expression of FoxO3a and increased expression of AMPK. Inhibition; of DNA-PK suppressed phosphorylation of Akt under nutrient starvation. Inhibition of DNA-PK or; Akt suppressed phosphorylations of MST1, FoxO3a, and NDR1 under nutrient starvation, which; suggests DNA-PKcs and Akt activate MST1, FoxO3a, and NDR1. Inhibition of DNA-PK did not; suppress phosphorylation ofAMPK under nutrient starvation. CONCLUSION: Our data suggest that DN-PKcs is activated under nutrient starvation and activates AktMST1, FoxO3a, and NDR1.