Rewiring E2F1 with classical NHEJ via APLF suppression promotes bladder cancer invasiveness

J Exp Clin Cancer Res. 2019 Jul 8;38(1):292. doi: 10.1186/s13046-019-1286-9.


Background: Bladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways.

Methods: RT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly.

Results: Herein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a 'high E2F1/APLF/DCLRE1C' signature, which was found to be particularly favorable for BC patient survival.

Conclusion: Suppression of the 'out-of-context' activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.

Keywords: APLF; Bladder cancer; E2F1; Non-homologous end-joining; miR-888.

MeSH terms

  • Cell Line, Tumor
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair*
  • DNA Methylation
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / antagonists & inhibitors
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / genetics
  • DNA-(Apurinic or Apyrimidinic Site) Lyase / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • E2F1 Transcription Factor / genetics
  • E2F1 Transcription Factor / metabolism*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Knockdown Techniques
  • Homologous Recombination
  • Humans
  • MicroRNAs / genetics
  • MicroRNAs / metabolism
  • Neoplasm Invasiveness
  • Poly-ADP-Ribose Binding Proteins / antagonists & inhibitors
  • Poly-ADP-Ribose Binding Proteins / genetics
  • Poly-ADP-Ribose Binding Proteins / metabolism*
  • Promoter Regions, Genetic
  • Transcriptional Activation
  • Urinary Bladder Neoplasms / genetics*
  • Urinary Bladder Neoplasms / metabolism*
  • Urinary Bladder Neoplasms / pathology


  • DNA-Binding Proteins
  • E2F1 Transcription Factor
  • E2F1 protein, human
  • MIRN888 microRNA, human
  • MicroRNAs
  • NHEJ1 protein, human
  • Poly-ADP-Ribose Binding Proteins
  • DCLRE1C protein, human
  • Endonucleases
  • APLF protein, human
  • DNA-(Apurinic or Apyrimidinic Site) Lyase
  • DNA Repair Enzymes