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

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, RN...

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Autores principales: Fletcher, C. E., Deng, L., Orafidiya, F., Yuan, W., Lorentzen, M. P. G. S., Cyran, O. W., Varela-Carver, A., Constantin, T. A., Leach, D. A., Dobbs, F. M., Figueiredo, I., Gurel, B., Parkes, E., Bogdan, D., Pereira, R. R., Zhao, S. G., Neeb, A., Issa, F., Hester, J., Kudo, H., Liu, Y., Philippou, Y., Bristow, R., Knudsen, K., Bryant, R. J., Feng, F. Y., Reed, S. H., Mills, I. G., de Bono, J., Bevan, C. L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8939142/
https://www.ncbi.nlm.nih.gov/pubmed/35317841
http://dx.doi.org/10.1186/s12943-022-01540-w
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author Fletcher, C. E.
Deng, L.
Orafidiya, F.
Yuan, W.
Lorentzen, M. P. G. S.
Cyran, O. W.
Varela-Carver, A.
Constantin, T. A.
Leach, D. A.
Dobbs, F. M.
Figueiredo, I.
Gurel, B.
Parkes, E.
Bogdan, D.
Pereira, R. R.
Zhao, S. G.
Neeb, A.
Issa, F.
Hester, J.
Kudo, H.
Liu, Y.
Philippou, Y.
Bristow, R.
Knudsen, K.
Bryant, R. J.
Feng, F. Y.
Reed, S. H.
Mills, I. G.
de Bono, J.
Bevan, C. L.
author_facet Fletcher, C. E.
Deng, L.
Orafidiya, F.
Yuan, W.
Lorentzen, M. P. G. S.
Cyran, O. W.
Varela-Carver, A.
Constantin, T. A.
Leach, D. A.
Dobbs, F. M.
Figueiredo, I.
Gurel, B.
Parkes, E.
Bogdan, D.
Pereira, R. R.
Zhao, S. G.
Neeb, A.
Issa, F.
Hester, J.
Kudo, H.
Liu, Y.
Philippou, Y.
Bristow, R.
Knudsen, K.
Bryant, R. J.
Feng, F. Y.
Reed, S. H.
Mills, I. G.
de Bono, J.
Bevan, C. L.
author_sort Fletcher, C. E.
collection PubMed
description 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12943-022-01540-w.
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spelling pubmed-89391422022-03-23 A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer Fletcher, C. E. Deng, L. Orafidiya, F. Yuan, W. Lorentzen, M. P. G. S. Cyran, O. W. Varela-Carver, A. Constantin, T. A. Leach, D. A. Dobbs, F. M. Figueiredo, I. Gurel, B. Parkes, E. Bogdan, D. Pereira, R. R. Zhao, S. G. Neeb, A. Issa, F. Hester, J. Kudo, H. Liu, Y. Philippou, Y. Bristow, R. Knudsen, K. Bryant, R. J. Feng, F. Y. Reed, S. H. Mills, I. G. de Bono, J. Bevan, C. L. Mol Cancer Research 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12943-022-01540-w. BioMed Central 2022-03-22 /pmc/articles/PMC8939142/ /pubmed/35317841 http://dx.doi.org/10.1186/s12943-022-01540-w Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
spellingShingle Research
Fletcher, C. E.
Deng, L.
Orafidiya, F.
Yuan, W.
Lorentzen, M. P. G. S.
Cyran, O. W.
Varela-Carver, A.
Constantin, T. A.
Leach, D. A.
Dobbs, F. M.
Figueiredo, I.
Gurel, B.
Parkes, E.
Bogdan, D.
Pereira, R. R.
Zhao, S. G.
Neeb, A.
Issa, F.
Hester, J.
Kudo, H.
Liu, Y.
Philippou, Y.
Bristow, R.
Knudsen, K.
Bryant, R. J.
Feng, F. Y.
Reed, S. H.
Mills, I. G.
de Bono, J.
Bevan, C. L.
A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer
title A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer
title_full A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer
title_fullStr A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer
title_full_unstemmed A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer
title_short A non-coding RNA balancing act: miR-346-induced DNA damage is limited by the long non-coding RNA NORAD in prostate cancer
title_sort non-coding rna balancing act: mir-346-induced dna damage is limited by the long non-coding rna norad in prostate cancer
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8939142/
https://www.ncbi.nlm.nih.gov/pubmed/35317841
http://dx.doi.org/10.1186/s12943-022-01540-w
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