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.

Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced ‘castrate-resistant’ disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition. Corresponding miRs increased AR activity through a novel and anti-dogmatic mechanism of direct association with AR 6.9 kb 3′UTR and transcript stabilisation. In addition, miR-346 and miR-361-3p modulation altered levels of constitutively active AR variants, and inhibited variant-driven PC cell proliferation, so may contribute to persistent AR signalling in CRPC in the absence of circulating androgens. Pathway analysis of AGO-PAR-CLIP-identified miR targets revealed roles in DNA replication and repair, cell cycle, signal transduction and immune function. Silencing these targets, including tumour suppressors ARHGDIA and TAGLN2, phenocopied miR effects, demonstrating physiological relevance. MiR-346 additionally upregulated the oncogene, YWHAZ, which correlated with grade, biochemical relapse and metastasis in patients. These AR-modulatory miRs and targets correlated with AR activity in patient biopsies, and were elevated in response to long-term enzalutamide treatment of patient-derived CRPC xenografts. In summary, we identified miRs that modulate AR activity in PC and CRPC, via novel mechanisms, and may represent novel PC therapeutic targets.

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.

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.

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.

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.