PLK1 Regulates MicroRNA Biogenesis through Drosha Phosphorylation.

Polo-Like Kinase 1 (PLK1), a key mediator of cell-cycle progression, is associated with poor prognosis and is a therapeutic target in a number of malignancies. Putative phosphorylation sites for PLK1 have been identified on Drosha, the main catalytic component of the microprocessor responsible for miR biogenesis. Several kinases, including GSK3ß, p70 S6 kinase, ABL, PAK5, p38 MAPK, CSNK1A1 and ANKRD52-PPP6C, have been shown to phosphorylate components of the miR biogenesis machinery, altering their activity and/or localisation, and therefore the biogenesis of distinct miR subsets. We hypothesised that PLK1 regulates miR biogenesis through Drosha phosphorylation. In vitro kinase assays confirmed PLK1 phosphorylation of Drosha at S300 and/or S302. PLK1 inhibition reduced serine-phosphorylated levels of Drosha and its RNA-dependent association with DGCR8. In contrast, a "phospho-mimic" Drosha mutant showed increased association with DGCR8. PLK1 phosphorylation of Drosha alters Drosha Microprocessor complex subcellular localisation, since PLK1 inhibition increased cytosolic protein levels of both DGCR8 and Drosha, whilst nuclear levels were decreased. Importantly, the above effects are independent of PLK1's cell cycle-regulatory role, since altered Drosha:DGCR8 localisation upon PLK1 inhibition occurred prior to significant accumulation of cells in M-phase, and PLK1-regulated miRs were not increased in M-phase-arrested cells. Small RNA sequencing and qPCR validation were used to assess downstream consequences of PLK1 activity on miR biogenesis, identifying a set of ten miRs (miR-1248, miR-1306-5p, miR-2277-5p, miR-29c-5p, miR-93-3p, miR-152-3p, miR-509-3-5p, miR-511-5p, miR-891a-5p and miR-892a) whose expression levels were statistically significantly downregulated by two pharmacological PLK1 kinase domain inhibitors, RO-5203280 and GSK461364. Opposingly, increased levels of these miRs were observed upon transfection of wild-type or constitutively active PLK1. Importantly, pre-miR levels were reduced upon PLK1 inhibition, and pri-miR levels decreased upon PLK1 activation, and hence, PLK1 Drosha phosphorylation regulates MiR biogenesis at the level of pri-miR-to-pre-miR processing. In combination with prior studies, this work identifies Drosha S300 and S302 as major integration points for signalling by several kinases, whose relative activities will determine the relative biogenesis efficiency of different miR subsets. Identified kinase-regulated miRs have potential for use as kinase inhibitor response-predictive biomarkers, in cancer and other diseases.

AAV9 intracerebroventricular gene therapy improves lifespan, locomotor function and pathology in a mouse model of Niemann-Pick type C1 disease.

Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.

A novel role for GSK3ß as a modulator of Drosha microprocessor activity and MicroRNA biogenesis.

Regulation of microRNA (miR) biogenesis is complex and stringently controlled. Here, we identify the kinase GSK3ß as an important modulator of miR biogenesis at Microprocessor level. Repression of GSK3ß activity reduces Drosha activity toward pri-miRs, leading to accumulation of unprocessed pri-miRs and reduction of pre-miRs and mature miRs without altering levels or cellular localisation of miR biogenesis proteins. Conversely, GSK3ß activation increases Drosha activity and mature miR accumulation. GSK3ß achieves this through promoting Drosha:cofactor and Drosha:pri-miR interactions: it binds to DGCR8 and p72 in the Microprocessor, an effect dependent upon presence of RNA. Indeed, GSK3ß itself can immunoprecipitate pri-miRs, suggesting possible RNA-binding capacity. Kinase assays identify the mechanism for GSK3ß-enhanced Drosha activity, which requires GSK3ß nuclear localisation, as phosphorylation of Drosha at S300 and/or S302; confirmed by enhanced Drosha activity and association with cofactors, and increased abundance of mature miRs in the presence of phospho-mimic Drosha. Functional implications of GSK3ß-enhanced miR biogenesis are illustrated by increased levels of GSK3ß-upregulated miR targets following GSK3ß inhibition. These data, the first to link GSK3ß with the miR cascade in humans, highlight a novel pro-biogenesis role for GSK3ß in increasing miR biogenesis as a component of the Microprocessor complex with wide-ranging functional consequences.

AR-v7 liquid biopsy for treatment stratification in prostate cancer: how close are we?

PURPOSE OF REVIEW: Recent clinical introduction of the novel antiandrogen, Enzalutamide (Enza), CYP17 inhibitor, Abiraterone (Abi), and the second-generation chemotherapeutic, Cabazitaxel, has increased survival of patients with advanced, metastatic castration-resistant prostate cancer (mCRPC). However, de novo and acquired resistance rates are high. A liquid biopsy that can rapidly, sensitively and robustly identify which patients will respond to treatment in a minimally invasive manner is urgently required to permit switch to a potentially more efficacious drug regimen, thus increasing survival whilst avoiding debilitating side effects associated with unnecessary treatment. This review will highlight recent developments in detection of AR-v7 in circulating mRNA/whole blood and circulating tumour cells (CTCs) as a liquid biopsy for patient-stratification in mCRPC. RECENT FINDINGS: Continued androgen receptor (AR) activity in mCRPC has been linked to the expression of a number of truncated but constitutively active AR isoforms. One such variant, AR-v7, can drive drug resistance in preclinical models and is correlated with disease progression whilst showing dynamic response to AR-targeting treatments when assessed in blood. It has thus been proposed as an Abi/Enza treatment-response biomarker. SUMMARY: AR-v7 liquid biopsy has the potential to transform clinical management of mCRPC and increase patient survival. This review will explore recent efforts to validate AR-v7 as a robust, clinically informative biomarker. I will also address potential limitations of detection and quantification that could frustrate its adoption into routine clinical practise.

Androgen-regulated processing of the oncomir miR-27a, which targets Prohibitin in prostate cancer.

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 modulatory miR-1271-5p can promote hormone sensitive prostate cancer cell growth.

In most patients with advanced prostate cancer treated with hormonal therapy, androgen independence eventually emerges, leading to death. Androgen receptor signalling remains an important prostate cancer driver, even in the advanced disease stage. MicroRNAs (miRs), non-coding RNAs that regulate gene expression by inhibiting translation and/or promoting degradation of target mRNAs, can act as tumour suppressors or "oncomiRs" and modulate tumour growth. Because of their stability in tissues and in circulation, and their specificity, microRNAs have emerged as potential biomarkers, as well as therapeutic targets in cancer. We identified miR-1271-5p as an androgen receptor modulatory microRNA and we show it can promote hormone sensitive prostate cancer cell growth. Inhibition or overexpression of miR-1271-5p levels affects prostate cancer cell growth, apoptosis and expression of both androgen receptor target genes and other genes that are likely direct targets, dependent on androgen receptor status, and tumour stage. We conclude that miR-1271-5p has the potential to drive progression of hormone-dependent disease and that the use of specific inhibitors of miR-1271-5p may have therapeutic potential in prostate cancer.

Costs of medical evacuation and transportation of First Nations Peoples and Inuit who travel for medical care in Canada: A systematic review.

OBJECTIVE: For First Nations people and Inuit who live on reserves or in rural and remote areas, a guideline requires their travel to urban centres once their pregnancy reaches 36-38 weeks gestation age to await labour and birth. While not encoded in Canadian legislation, this guideline-and invisible policy-is reinforced by the lack of alternatives. Research has repeatedly demonstrated the harm of obstetric evacuation, causing emotional, physical, and financial stress for pregnant and postpartum Indigenous women and people. Our objective was to describe the costs of obstetric evacuation, as reported in the literature. METHODS: We conducted a systematic review using online searches of electronic databases (Ovid EMBASE, CINAHL, Ovid Healthstar, PubMed, ScienceDirect, PROSPERO, and Cochrane Database of Systematic Reviews) and identified studies that reported costs related to medical evacuation or transportation in rural and remote Indigenous communities. We performed critical appraisal of relevant studies. SYNTHESIS: We identified 19 studies that met the inclusion criteria. The studies reported various types of cost, including direct, indirect, and intangible costs. Medical evacuation costs ranged from CAD $7714 to CAD $31,794. Indirect and intangible costs were identified, including lost income and lack of respect for cultural practices. CONCLUSION: Costs associated with obstetric evacuation are high, with medical evacuation as the most expensive direct cost identified. Although we were able to identify a range of costs, information on financing and funding flows was unclear. Across Canada, additional research is required to understand the direct costs of obstetric evacuation to Indigenous Peoples and communities.

RéSUMé: OBJECTIF: Une ligne directrice oblige les personnes inuites et des Premières Nations vivant dans des réserves ou des régions rurales et isolées et qui en sont entre leur 36e et leur 38e semaine de grossesse à se rendre dans un centre urbain pour y attendre le travail et l'accouchement. Bien qu'elle ne soit pas enchâssée dans la loi canadienne, cette ligne directrice (et le principe qu'elle cache) est renforcée par l'absence de solutions de rechange. Des études ont démontré à maintes reprises les préjudices de l'évacuation obstétricale, qui cause un stress émotionnel, physique et financier pour les femmes et les personnes enceintes autochtones en période postpartum. Nous avons cherché à décrire les coûts de l'évacuation obstétricale figurant dans la littérature spécialisée. MéTHODE: Nous avons mené une revue systématique en consultant des bases de données électroniques (Ovid EMBASE, CINAHL, Ovid Healthstar, PubMed, ScienceDirect, PROSPERO et Cochrane Database of Systematic Reviews), puis en répertoriant les études faisant état des coûts de l'évacuation médicale ou du transport médical dans les communautés autochtones rurales et éloignées. Nous avons ensuite effectué une évaluation critique des études pertinentes. SYNTHèSE: Dix-neuf études répondaient aux critères d'inclusion. Elles faisaient état de divers types de coûts : directs, indirects et intangibles. Les coûts de l'évacuation médicale variaient de 7 714 $ à 31 794 $ CAN. Les coûts indirects et intangibles identifiés étaient la perte de revenu et le manque de respect pour les pratiques culturelles. CONCLUSION: Les coûts associés à l'évacuation obstétricale sont importants, et le coût direct le plus élevé est celui de l'évacuation médicale. Nous avons été en mesure de cerner une fourchette de coûts, mais les informations sur le financement et les flux de financement n'étaient pas claires. Partout au Canada, il faudrait pousser la recherche pour connaître les coûts directs de l'évacuation obstétricale pour les personnes et les communautés autochtones.

Crosstalk between Long Non Coding RNAs, microRNAs and DNA Damage Repair in Prostate Cancer: New Therapeutic Opportunities?

It is increasingly appreciated that transcripts derived from non-coding parts of the human genome, such as long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), are key regulators of biological processes both in normal physiology and disease. Their dysregulation during tumourigenesis has attracted significant interest in their exploitation as novel cancer therapeutics. Prostate cancer (PCa), as one of the most diagnosed malignancies and a leading cause of cancer-related death in men, continues to pose a major public health problem. In particular, survival of men with metastatic disease is very poor. Defects in DNA damage response (DDR) pathways culminate in genomic instability in PCa, which is associated with aggressive disease and poor patient outcome. Treatment options for metastatic PCa remain limited. Thus, researchers are increasingly targeting ncRNAs and DDR pathways to develop new biomarkers and therapeutics for PCa. Increasing evidence points to a widespread and biologically-relevant regulatory network of interactions between lncRNAs and miRNAs, with implications for major biological and pathological processes. This review summarises the current state of knowledge surrounding the roles of the lncRNA:miRNA interactions in PCa DDR, and their emerging potential as predictive and diagnostic biomarkers. We also discuss their therapeutic promise for the clinical management of PCa.

An Environmental Scan of Indigenous Cultural Safety in Canadian Baccalaureate Nursing and Midwifery Programs.

BACKGROUND: The Truth and Reconciliation Commission (TRC) TRC has called to increase the number of Indigenous practitioners and include cultural competency education in their curricula. However, it remains unknown how nursing and midwifery programs are progressing towards these goals. PURPOSE: To examine the extent to which baccalaureate nursing and midwifery programs are creating culturally safe spaces for Indigenous students, responding to TRC-recommended curricular changes, and including Indigenous content. METHODS: A digital environmental scan of accredited baccalaureate nursing and midwifery programs in Canada was conducted. Analysis was conducted using descriptive statistics. RESULTS: Of the 107 programs, less than one-fifth (n = 19, 17.8%) met all three cultural safety criteria. More than half (n = 59, 55.1%) included culturally safe spaces for Indigenous students, 20 (18.7%) satisfied TRC call #24 to require Indigenous-relevant coursework, and one-third (n = 36, 33.6%) were seen as infusing their curricula with Indigenous-related content. CONCLUSIONS: This represents the first attempt to systematically catalog nursing and midwifery programs' response to the TRC Calls to Action. Most schools have not made substantial progress towards cultural safety. Nursing and midwifery programs should commit to expanding their cultural safety programming to incorporate multiple ways of knowing and being in their curricula.

A call for action that cannot go to voicemail: Research activism to urgently improve Indigenous perinatal health and wellbeing.

In this call to action, a coalition of Indigenous and non-Indigenous researchers from Australia, Aotearoa New Zealand, United States and Canada argue for the urgent need for adequately funded Indigenous-led solutions to perinatal health inequities for Indigenous families in well-resourced settler-colonial countries. Authors describe examples of successful community-driven programs making a difference and call on all peoples to support and resource Indigenous-led perinatal health services by providing practical actions for individuals and different groups.

Coordinated AR and microRNA regulation in prostate cancer.

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.

Androgen receptor-modulatory microRNAs provide insight into therapy resistance and therapeutic targets in advanced 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.

An assessment of GafChromic film for measuring 50 kV and 100 kV percentage depth dose curves.

Percentage depth dose (PDD) curves were obtained for 50 kV and 100 kV x-rays on a Gulmay Medical D3000 DXR unit. Different dosimetry systems were compared including a Scanditronix Wellhofer small volume cylindrical ion chamber, a Wellhofer photon PFD diode, a PTW soft x-ray parallel plate chamber (N23342) and two types of radiochromic film: GafChromic EBT and GafChromic MD55. The PDD curves were also compared to BEAMnrc Monte Carlo predictions. GafChromic film was found to be a valid choice of dosimeter for measuring percentage depth dose curves at 100 kV and 50 kV. All the dosimeters showed agreement with predictions at depths greater than 10 mm, while near the surface GafChromic film and PFD diodes give the best agreement to Monte Carlo values.

College students and HIV/AIDS: a comparison of nontraditional and traditional student perspectives.

OBJECTIVE AND PARTICIPANTS: The authors compared nontraditional college students' knowledge and perceptions of HIV/AIDS and sexual practices with previously reported results about traditional students. METHODS: Nontraditional students completed an online survey with questions based on national HIV/AIDS surveys. Traditional students completed the same survey with paper and pencil. RESULTS: Overall, the authors found more similarities than differences between the 2 groups. The findings support previous research that suggests that although college students are knowledgeable about HIV and its risks, they express little personal concern about becoming infected. The authors also discuss how apparent differences between nontraditional and traditional students regarding personal concern about becoming infected, relationship status, and information sources may influence the development of effective prevention strategies geared toward nontraditional college students. CONCLUSIONS: Institutional leaders need to adapt to these differences, and researchers should undertake additional studies to clarify these differences so that college students may be more effectively educated about HIV/AIDS and encouraged to get tested.

The relationship between knowledge of pain neurophysiology and fear avoidance in people with chronic pain: A point in time, observational study.

Chronic pain is prevalent in the western world; however fear of pain often has a greater impact than the degree of initial injury. The aim of this study was to explore the relationship between knowledge of the neurophysiology of pain and fear avoidance in individuals diagnosed with chronic pain. Twenty-nine people with chronic musculoskeletal pain were recruited and completed questionnaires to determine their understanding of pain neurophysiology and the degree of their fear avoidance beliefs. There was an inverse relationship between knowledge of pain neurophysiology and the level of fear avoidance. Patients with higher pain knowledge reported less fear avoidance and lower perceived disability due to pain. There was no relationship with the educational level or compensable status for either variable. The findings suggest that fear avoidance is positively influenced by neurophysiology of pain education, so that a higher level of pain knowledge is associated with less activity-related fear. The clinical implication is that reducing fear avoidance/kinesiophobia using neurophysiology of pain education in people with chronic pain may provide an effective strategy to help manage fear avoidance and related disability in the chronic pain population in order to improve treatment outcomes.

Interplay between steroid signalling and microRNAs: implications for hormone-dependent cancers.

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.

Circulating nucleic acids as biomarkers of prostate cancer.

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.

Circulating peripheral blood mononuclear cells exhibit altered miRNA expression patterns in pancreatic 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.