Abstract
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.
Highlights
Prostate cancer (PC) is the most prevalent malignancy in Western males [1]
We further investigated the ability of miR-346, 1361-3p and -197 to regulate proliferation of androgen receptor (AR) variant-driven PC cell lines, by comparing proliferation between parental 22RV1 cells and WTknock out derivatives (22RV1-AR-FL-KO), and between parental CWR-R1-AD1 cells and the CWR-R1-D567es derivative lacking WT-AR but overexpressing v567es
To provide further evidence to support the rationale of combined miR-346, -361-3p or -197 inhibitor and antiandrogen treatment, we investigated the effects of ARmodulatory miR manipulation on apoptotic and proliferative response to the anti-androgen, Enzalutamide (Enza), in PC cells
Summary
Growth of prostate tumours is dependent on circulating androgens activating the androgen receptor (AR) [2]. Targets of this ligand-activated transcription factor include effectors of cell cycle progression and proliferation, androgen deprivation remains a first-line approach for treatment of metastatic PC. Despite the lack of requirement of CRPC tumours for circulating androgens, it is widely accepted that AR signalling is still driving their growth. Mechanisms of persistent AR activity include increased sensitivity to remaining androgens (e.g. adrenal androgens), ligand promiscuity, AR gene amplification or mutation, altered AR mRNA splicing leading to constitutively active forms, aberrant expression of AR coregulators and intra-tumoural androgen synthesis [3]. Acquired resistance to drugs targeting the AR ligand binding domain (LBD) is an increasing clinical problem, so novel therapeutics that repress AR through non-LBD-mediated mechanisms will be important additions to the CRPC treatment repertoire
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