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

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

Author information

1
Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Schillingallee 69, 18057, Rostock, Germany.
2
Institute of Medical Radiation Biology, University of Duisburg-Essen Medical School, Essen, Germany.
3
Institute of Experimental Gene Therapy and Cancer Research, Rostock University Medical Center, Schillingallee 69, 18057, Rostock, Germany. brigitte.puetzer@med.uni-rostock.de.
4
Department Life, Light and Matter of the Interdisciplinary Faculty at Rostock University, Rostock, Germany. brigitte.puetzer@med.uni-rostock.de.

Abstract

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

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