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NORAD (NOn-Coding RNA Activated by DNA Damage) is a highly-abundant, evolutionarily-conserved lncRNA. It maintains mitosis, DNA damage repair (DDR), and chromosomal integrity through PUM1/2 sequestration (PUM1/2 activity increases turnover of DDR factors), and through formation of a TOPO2-containing complex critical for genome integrity. We show that NORAD activity is regulated by microRNA-346 (miR-346), which disrupts NORAD:PUM2, interaction, leading to PUM2 destabilization and derepression of PUM1/2 DDR targets in prostate cancer (PCa) cells. RNA-seq reveals widespread miR-346 dysregulation of DNA damage, DNA replication and cell cycle transcripts. A novel method for high resolution, amplification-free genome-wide mapping of double strand DNA breaks (DSBs) (INDUCE-seq) reveals miR-346 induces DSBs specifically at transcription start sites characterized by phospho-PolII/CTCF/ZFX binding - a phenomenon not previously described for any microRNA. Mechanistically, DSBs result from miR-346 activation of transcription, R-loop formation and replicative catastrophe. This results in rapid dose-dependent induction of DNA damage, leading to checkpoint activation and cytosolic DNA accumulation, rescuable by NORAD. This cytosolic DNA activates cytokine-inducing cGAS-STING/RIG-1 innate immune pathways. Indeed, RNA-seq analysis reveals the top NORAD-enriched pathway as interferon signaling, while cytokine arrays reveal secretion of pro-Treg, MDSC and TAM factors by NORAD-overexpressing cells. NORAD inversely correlates with tumor immune response in gene expression data sets, and expression-based immune infiltration scoring predicts increased M2 macrophages, and reduced NK, CD8+ve, Th1 and cytotoxic T cells in NORAD-high vs NORAD-low PCa, indicating an ‘immune-cold' microenvironment. Excitingly, NORAD silencing results in several thousand-fold increase in mature miR-346 without affecting pri-miR levels, supporting NORAD's ability to drive target-directed microRNA decay (TDMD) of miR-346 as a critical novel genome protection mechanism. However, miR-346-induced DNA damage is in part NORAD-independent, since miR-346 induces DSBs within 1h, and in contrast to most miRs is predominantly chromatin-bound (NORAD is cytoplasmic). Critically, miR-346 sensitizes PCa cells to DNA-damaging chemotherapy and PARP inhibition. MiR-346 expression is associated with improved PCa survival, and reduced in high vs low Gleason grade PCa. Notably, NORAD strongly correlates with DDR signatures in early-stage, but not advanced metastatic PCa. Despite its DDR-promoting activity, and in contrast to miR-346, it is associated with worse survival across multiple patient cohorts. In conclusion, these data demonstrate that the NORAD:miR-346 interaction determines DNA damage response and innate immune pathway activity to regulate tumor immune response in PCa. Citation Format: Claire Fletcher, Lin Deng, Folake Orafidiya, Wei Yuan, Ines Figueiredo, Bora Gurel, Damien Leach, Fadi Issa, Antje Neeb, Denisa Bogdan, Felix Dobbs, Yiannis Philippou, Emma A. Murphy, Shuang G. Zhao, Joanna Hester, Richard J. Bryant, Simon H. Reed, Karen E. Knudsen, Ian G. Mills, Johann de Bono, Charlotte L. Bevan. Long non-coding RNA NORAD interaction with miR-346 impacts DNA damage response and anti-tumor immunity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2362.

The androgen receptor (AR) remains a key driver of prostate cancer (PCa) progression, even in the advanced castrate-resistant stage, where testicular androgens are absent. It is therefore of critical importance to understand the molecular mechanisms governing its activity and regulation during prostate tumourigenesis. MicroRNAs (miRs) are small ∼22 nt non-coding RNAs that regulate target gene, often through association with 3′ untranslated regions (3′UTRs) of transcripts. They display dysregulation during cancer progression, can function as oncogenes or tumour suppressors, and are increasingly recognised as targets or regulators of hormonal action. Thus, understanding factors which modulate miRs synthesis is essential. There is increasing evidence for complex and dynamic bi-directional cross-talk between the multi-step miR biogenesis cascade and the AR signalling axis in PCa. This review summarises the wealth of mechanisms by which miRs are regulated by AR, and conversely, how miRs impact AR's transcriptional activity, including that of AR splice variants. In addition, we assess the implications of the convergence of these pathways on the clinical employment of miRs as PCa biomarkers and therapeutic targets.

193 Background: Prostate cancer (PCa) is an androgen dependent malignancy. PCa patients initially respond to androgen ablation but eventually the disease becomes castrate resistant. MicroRNAs (miRs) are considered to be master regulators of gene expression and could be potential biomarkers and therapeutic targets for PCa. We previously showed miR-27a-3p to be an androgen receptor (AR)-modulatory oncomiR, with a role in PCa progression. Methods: RNA sequencing was carried out on xenograft tumours treated with an inhibitory antisense oligonucleotide for miR-27a (ASO-27a), or control. Differentially expressed genes were cross-referenced with publicly available patient tissue datasets. ASO-27a was transfected in a panel of PCa cell lines mirroring disease progression, to verify changes in transcriptional and translational levels. Immunohistochemical analysis of encoded proteins was undertaken in a cohort of 46 PCa patients with Gleason 3+4 or 4+3 disease and compared with normal tissue-control. Results: Putative target genes tested included the tumour suppressor HIP2, the oncogenes SRC3, YAP, and PAX3, which can act both as oncogene and tumour suppressor. MRNA or protein levels of the majority of those genes tested were upregulated after ASO-27a treatment of PCa cell lines. In 22Rv1 cells, HIPK2 mRNA levels were significantly upregulated with ASO-27a (p = 0.0137), while protein levels were downregulated . PAX3 mRNA levels were significantly reduced by ASO-27a in 22Rv1 cells (p = 0.0364), while protein levels were significantly increased (p = 0.0112). Immunohistochemically, PAX3 protein was lower in Gleason 7 tumours than in normal tissue, while YAP expression was higher in patients with Gleason 4+3, relative to those with 3+4. SRC3 immunostaining was slightly, but not significantly, higher in patients with 3+4 compared to 4+3. Conclusions: MiR-27a-3p is an AR-modulatory oncomiR in PCa, with potential as a therapeutic target. MiR-27a interaction with its target genes and especially HIPK2, YAP, SRC3 and PAX3 could provide the basis for therapeutic advance in screening and in the treatment of castrate resistant disease, through ASO-27a inhibition.

Hormones are key drivers of cancer development. To date, interest has largely been focussed on the classical model of hormonal gene regulation, but there is increasing evidence for a role of hormone signalling pathways in post-translational regulation of gene expression. In particular, a complex and dynamic network of bi-directional interactions with microRNAs (miRs) at all stages of biogenesis and during target gene repression is emerging. miRs, which act mainly by negatively regulating gene expression through association with 3′-UTRs of mRNA species, are increasingly understood to be important in development, normal physiology and pathogenesis. Given recent demonstrations of altered miR profiles in a diverse range of cancers, their ability to function as oncogenes or tumour suppressors, and hormonal regulation of miRs, understanding mechanisms by which miRs are generated and regulated is vitally important. miRs are transcribed by RNA polymerase II and then processed in the nucleus by the Drosha-containing Microprocessor complex and in the cytoplasm by Dicer, before mature miRs are incorporated into the RNA-induced silencing complex. It is increasingly evident that multiple cellular signalling pathways converge upon the miR biogenesis cascade, adding further layers of regulatory complexity to modulate miR maturation. This review summarises recent advances in identification of novel components and regulators of the Microprocessor and Dicer complexes, with particular emphasis on the role of hormone signalling pathways in regulating their activity. Understanding hormone regulation of miR production and how this is perturbed in cancer are critical for the development of miR-based therapeutics and biomarkers.

Introduction Prostate cancer (PCa) is the most common type of cancer affecting the male population. It is an androgen dependent malignancy that initially responds well to androgen ablation therapy. However, despite hormonal treatment, castrate resistant prostate cancer eventually emerges. MicroRNAs (miRs), a class of small non-coding RNAs, modulate gene silencing through inhibition of translation and mRNA degradation. MiRs and/or miR inhibitors that reduce AR activity represent a promising therapeutic strategy, therefore a high-throughput miR inhibitor screen was performed in PCa cell lines, expressing a luciferase-based AR reporter. We sought to validate and characterise AR-modulatory miRs and identify their targets in PCa cell lines. Material and methods Prostate cancer cell lines that stably expressed an AR reporter element were transfected with specific miR inhibitors and mimics. The effect of miR modulation on potential alteration of AR activity was investigated (through a luciferase assay), as well as the potential impact on cell growth by an SRB assay and on apoptosis, using a caspase 3/7 assay. Real-time qPCR and western blot assays were performed, to check the potential effect of miR manipulation on mRNA and protein levels of AR and miR specific target genes. Results and discussions MiR-1271–5 p inhibitor significantly reduced AR activity and increased apoptosis in a castrate resistant cell line and the opposite effect was seen with the use of the mimic. MiR-1271–5 p inhibitor significantly reduced growth of the AR dependent cell line and miR-1271–5 p mimic had the opposite effect. AR mRNA levels were altered, as well as levels of miR specific target genes, after manipulation of PCa cell lines with miR-1271–5 p inhibitor and mimic. Similar effects were noticed with the use of an antisense oligonucleotide for miR-27a-3p, which has been previously demonstrated to be an AR regulated oncomiR in PCa. Conclusion From a panel of different miRs, we have demonstrated the androgenic potential and oncogenic capacity of miR-1271–5 p in PCa. Also, the use of antisense oligonucleotides for inhibition of miR-27a-3p has therapeutic potential for castrate resistant disease. Predicted targets of both miR-27a-3p and miR-1271–5 p include genes with roles in growth arrest, apoptosis and DNA repair. Further studies are ongoing, to elucidate the molecular and biological role of these miRs in PCa, providing a better understanding of disease development, as well as a foundation for advances in diagnosis and in treatment.

e16562 Background: Current screening methodologies for prostate cancer (PCa) are relatively insensitive and there is a need for new treatments for castrate resistant disease. MicroRNAs (miRs) are considered to be master regulators of the genome. We have investigated the role of miRs in modulating androgen receptor function and their potential as treatments of PCa. We report that the AR-modulatory miR-1271-5p also targets SND1 and MORF4L1 and may have a role in PCa progression and screening. Methods: AGO2-PAR-CLIP analysis was performed to ascertain miR-1271-5p target genes in human PCa cell lines. MiR-1271-5p levels were manipulated in cell lines by transfection with miR mimic and or antisense inhibitor. SND1 and MORF4L1 were confirmed as targets by real-time qPCR and western blotting. The functional role of miR-1271-5p and its target genes was assessed by SRB growth assays . Immunohistochemical detection of SND1 and MORF4L1 expression was studied in a cohort of 63 PCa patients and compared with normal controls. Results: MORF4L1 mRNA levels were significantly reduced with the use of miR-1271-5p mimic in 22RV1 cells (p = 0.001), while SND1 mRNA levels were significantly decreased with the use of miR-1271-5p inhibitor in C42 cells (p = 0.0014). Targeting SND1 or MORF4L1, in combination with miR inhibition, significantly reduced C42 (p = 0.0003) and 22RV1 (p = 0.0014) cell growth. MORF4L1 expression was higher in patients with Gleason score (GS) 4+3 relative to those with GS 3+4 and in PCa tissue, as compared with normal prostatic tissue, but did not reach significance. SND1 immunostaining was significantly higher in patients with GS 4+3 or GS 3+4 PCa, compared with normal prostatic tissue (p = 0.0211, p = 0.0007 respectively). SND1 staining was significantly higher in patients with GS 4+3, compared to GS 3+4 (p = 0.0431) or GS 3+3 (p = 0.0251). Conclusions: MiR-1271-5p is an AR-modulatory microRNA, which shows great potential as a biomarker and therapeutic target in PCa. The interaction of miR-1271-5p with its target genes SND1 and MORF4L1 could provide the basis for therapeutic advance in screening and in the treatment of castrate resistant PCa.

Prostate cancer, the most common cancer of western men, requires new biomarkers, especially given that the benefits of PSA testing remain uncertain. Nucleic acids can now be accurately and sensitively detected in human blood. Over the last decade, investigations into utility of circulating cell-free miRNA, DNA and mRNA as novel biomarkers have expanded exponentially. In the near future, they may be routinely used to accurately diagnose cancers, stratify indolent from aggressive disease and inform treatment decisions. However, advancement of such tests into clinical settings is hampered by technical problems with assay specificity and sensitivity, and small study sizes. This review highlights the different forms of circulating nucleic acids and those that show the most potential as viable biomarkers for prostate cancer.

Evaluation of: Wang WS, Liu LX, Li GP et al. Combined serum CA19-9 and miR-27a-3p in peripheral blood mononuclear cells to diagnose pancreatic cancer. Cancer Prev. Res. (Phila.) 6(4), 331-338 (2013). Patients with pancreatic ductal adenocarcinoma (PDAC) have a bleak outlook, primarily because tumors are detected late and are often too advanced for surgical resection. In addition, these lesions are incredibly resistant to anticancer therapies. The majority of PDAC patients have impaired tumor immunity, contributing to disease development and progression, although the mechanisms remain poorly understood. miRNAs are important negative gene regulators that have critical roles in human tumorigenesis. Blood-based miRNAs have been investigated as biomarkers for various cancers, in the hope that these will outperform current serum tumor markers. The evaluated study examined the miRNA profiles in peripheral blood mononuclear cells from PDAC patients. The theory is that circulating blood cells monitor the patients' physiological state and respond by altering their transcriptome and that this can then be used to detect disease. In this article, we have examined the evidence for using circulating miRNAs to diagnose/prognose PDAC.

MicroRNAs (miRs) play an important role in the development of many complex human diseases and may have tumour suppressor or oncogenic (oncomir) properties. Prostate cancer is initially an androgen-driven disease, and androgen receptor (AR) remains a key driver of growth even in castration-resistant tumours. However, AR-mediated oncomiR pathways remain to be elucidated. We demonstrate that miR-27a is an androgen-regulated oncomir in prostate cancer, acting via targeting the tumour suppressor and AR corepressor, Prohibitin (PHB). Increasing miR-27a expression results in reduced PHB mRNA and protein levels, and increased expression of AR target genes and prostate cancer cell growth. This involves a novel mechanism for androgen-mediated miR regulation, whereby AR induces a transient increase in miR-23a27a24-2 transcription, but more significantly accelerates processing of the primiR-23a27a24-2 cluster. Androgens therefore regulate miR-27a expression both transcriptionally (via AR binding to the cluster promoter) and post-transcriptionally (accelerating primiR processing to the mature form). We further show that a miR-27a anti-sense oligonucleotide, by opposing the effects of mir-27a, has therapeutic potential in prostate cancer.