A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer

Abstract

Background miR-346 was identified as an activator of Androgen Receptor (AR) signalling that associates with DNA damage response (DDR)-linked transcripts in prostate cancer (PC). We sought to delineate the impact of miR-346 on DNA damage, and its potential as a therapeutic agent. Methods RNA-IP, RNA-seq, RNA-ISH, DNA fibre assays, in vivo xenograft studies and bioinformatics approaches were used alongside a novel method for amplification-free, single nucleotide-resolution genome-wide mapping of DNA breaks (INDUCE-seq). Results miR-346 induces rapid and extensive DNA damage in PC cells - the first report of microRNA-induced DNA damage. Mechanistically, this is achieved through transcriptional hyperactivation, R-loop formation and replication stress, leading to checkpoint activation and cell cycle arrest. miR-346 also interacts with genome-protective lncRNA NORAD to disrupt its interaction with PUM2, leading to PUM2 stabilisation and its increased turnover of DNA damage response (DDR) transcripts. Confirming clinical relevance, NORAD expression and activity strongly correlate with poor PC clinical outcomes and increased DDR in biopsy RNA-seq studies. In contrast, miR-346 is associated with improved PC survival. INDUCE-seq reveals that miR-346-induced DSBs occur preferentially at binding sites of the most highly-transcriptionally active transcription factors in PC cells, including c-Myc, FOXA1, HOXB13, NKX3.1, and importantly, AR, resulting in target transcript downregulation. Further, RNA-seq reveals widespread miR-346 and shNORAD dysregulation of DNA damage, replication and cell cycle processes. NORAD drives target-directed miR decay (TDMD) of miR-346 as a novel genome protection mechanism: NORAD silencing increases mature miR-346 levels by several thousand-fold, and WT but not TDMD-mutant NORAD rescues miR-346-induced DNA damage. Importantly, miR-346 sensitises PC cells to DNA-damaging drugs including PARP inhibitor and chemotherapy, and induces tumour regression as a monotherapy in vivo, indicating that targeting miR-346:NORAD balance is a valid therapeutic strategy. Conclusions A balancing act between miR-346 and NORAD regulates DNA damage and repair in PC. miR-346 may be particularly effective as a therapeutic in the context of decreased NORAD observed in advanced PC, and in transcriptionally-hyperactive cancer cells.

LncRNA weighted gene co-expression network analysis reveals novel biomarkers related to prostate cancer metastasis

“…Significantly, regulation of NORAD affects the PUM proteins and changes the extracellular vesicle (EV) proteins that participate in communication between cells in the tumor microenvironment [ 52 , 53 ]. The effects of NORAD are implicated in almost all aspects of tumors, including carcinogenesis proliferation, apoptosis, invasion, and metastasis [ 54 ]. However, the precise molecular mechanisms of NORAD are still at a preliminary stage that requires further systematic investigation.…”

LncRNA weighted gene co-expression network analysis reveals novel biomarkers related to prostate cancer metastasis

“…Signi cantly, regulation of NORAD affects the PUM proteins and changes the extracellular vesicle (EV) proteins that participate in communication between cells in the tumor microenvironment [52,53]. The effects of NORAD are implicated in almost all aspects of tumors, including carcinogenesis proliferation, apoptosis, invasion, and metastasis [54]. However, the precise molecular mechanisms of NORAD are still at a preliminary stage that requires further systematic investigation.…”

PLK1 Regulates MicroRNA Biogenesis through Drosha Phosphorylation

“…Western blotting was performed as described [ 34 ]. Primary antibodies were rabbit anti-Drosha mAb (ab183732, Abcam, Cambridge, UK), mouse anti-PLK1 mAb (#4513, Cell Signalling, Danvers, MA, USA), mouse anti-Flag epitope tag M2 mAb (F1804, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-DGCR8 mAb (ab191875, Abcam), rabbit anti-GAPDH mAb (#2118, Cell Signalling), rabbit anti-Lamin B1 mAb (ab229025, Abcam), mouse anti-phospho-serine (P5747, Sigma-Aldrich) or mouse anti-β-actin mAb (ab6276, Abcam).…”