Ionizing radiation (IR), a prototypical DNA damaging agent, induces DNA double-strand breaks (DSBs). IR-induced DNA-DSBs are predominantly repaired by the non-homologous end joining (NHEJ) pathway. IR-induced DNA damage has been shown to induce autophagy, an intracellular degradation process that delivers cytoplasmic components to the lysosome. Here, we examined the interplay between autophagy and DNA damage response (DDR), with a particular emphasis on an E3 ubiquitin ligase RNF168, which is negatively regulated by a deubiquitinase (DUB) enzyme, USP14. Akt, a serine/ threonine protein kinase, is known to mediate the phosphorylation of USP14 on Ser432. Loss of the tumor suppressor PTEN, phosphatase and tensin homolog, results in hyperactivated Akt signaling, commonly reported in human malignancies, including prostate cancer (PCa).
Autophagy inhibition was coupled to diminished RNF168-mediated histone H2A ubiquitination. The E3 ubiquitin ligase RNF168 is essential for recruitment of the DNA-DSBs signaling proteins, including 53BP1, a critical DDR signaling effector at the DNA-DSBs sites. IR induced sustained ¿-H2AX, and reduced 53BP1 in autophagy-deficient PCa cells. Autophagy-deficient cells showed also reduced activity for DNA-PKcs, a kinase that is required for the NHEJ repair pathway. A defect in recruitment of DNA repair proteins could be caused by hyperactivity of the deubiquitinase USP14.
In response to IR, we show that USP14 was upregulated and generated foci in autophagy-deficient cells. Importantly, inhibiting autophagy following radiation-induced recruitment of USP14 onto chromatin. There was an inverse correlation between USP14 and RNF168 expression in autophagy-deficient PCa and non-small cell lung cancer (NSCLC) cells, indicating that USP14 may contribute to the aberrant inhibition of the NHEJ-DDR signaling pathway by targeting RNF168. USP14 inhibition by IU1, an enzymatic activity inhibitor or Akt inhibition rescued the activity of NHEJ-DDR proteins in autophagy-deficient cells. These findings uncovered a novel negative NHEJ regulation mechanism by USP14, which provided unique insights into the link between autophagy and NHEJ-DDR.