p62-mediated Selective autophagy endows virus-transformed cells with insusceptibility to DNA damage under oxidative stress

PLoS Pathog. 2019 Apr 24;15(4):e1007541. doi: 10.1371/journal.ppat.1007541. eCollection 2019 Apr.

Abstract

DNA damage response (DDR) and selective autophagy both can be activated by reactive oxygen/nitrogen species (ROS/RNS), and both are of paramount importance in cancer development. The selective autophagy receptor and ubiquitin (Ub) sensor p62 plays a key role in their crosstalk. ROS production has been well documented in latent infection of oncogenic viruses including Epstein-Barr Virus (EBV). However, p62-mediated selective autophagy and its interplay with DDR have not been investigated in these settings. In this study, we provide evidence that considerable levels of p62-mediated selective autophagy are spontaneously induced, and correlate with ROS-Keap1-NRF2 pathway activity, in virus-transformed cells. Inhibition of autophagy results in p62 accumulation in the nucleus, and promotes ROS-induced DNA damage and cell death, as well as downregulates the DNA repair proteins CHK1 and RAD51. In contrast, MG132-mediated proteasome inhibition, which induces rigorous autophagy, promotes p62 degradation but accumulation of the DNA repair proteins CHK1 and RAD51. However, pretreatment with an autophagy inhibitor offsets the effects of MG132 on CHK1 and RAD51 levels. These findings imply that p62 accumulation in the nucleus in response to autophagy inhibition promotes proteasome-mediated CHK1 and RAD51 protein instability. This claim is further supported by the findings that transient expression of a p62 mutant, which is constitutively localized in the nucleus, in B cell lines with low endogenous p62 levels recaptures the effects of autophagy inhibition on CHK1 and RAD51 protein stability. These results indicate that proteasomal degradation of RAD51 and CHK1 is dependent on p62 accumulation in the nucleus. However, small hairpin RNA (shRNA)-mediated p62 depletion in EBV-transformed lymphoblastic cell lines (LCLs) had no apparent effects on the protein levels of CHK1 and RAD51, likely due to the constitutive localization of p62 in the cytoplasm and incomplete knockdown is insufficient to manifest its nuclear effects on these proteins. Rather, shRNA-mediated p62 depletion in EBV-transformed LCLs results in significant increases of endogenous RNF168-γH2AX damage foci and chromatin ubiquitination, indicative of activation of RNF168-mediated DNA repair mechanisms. Our results have unveiled a pivotal role for p62-mediated selective autophagy that governs DDR in the setting of oncogenic virus latent infection, and provide a novel insight into virus-mediated oncogenesis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Autophagy*
  • Burkitt Lymphoma / genetics
  • Burkitt Lymphoma / metabolism
  • Burkitt Lymphoma / pathology
  • Burkitt Lymphoma / virology
  • Cell Transformation, Viral*
  • Chromatin
  • DNA Damage*
  • DNA Repair
  • Epstein-Barr Virus Infections / genetics
  • Epstein-Barr Virus Infections / metabolism
  • Epstein-Barr Virus Infections / pathology*
  • Epstein-Barr Virus Infections / virology
  • Herpesvirus 4, Human / physiology
  • Humans
  • Kelch-Like ECH-Associated Protein 1 / genetics
  • Kelch-Like ECH-Associated Protein 1 / metabolism
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism
  • Oxidative Stress*
  • Proteasome Endopeptidase Complex
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Reactive Oxygen Species / metabolism*
  • Signal Transduction
  • Ubiquitin / metabolism
  • Virus Latency

Substances

  • Chromatin
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • P62 protein, human
  • RNA-Binding Proteins
  • Reactive Oxygen Species
  • Ubiquitin
  • Proteasome Endopeptidase Complex