A systematic review reveals conflicting evidence for the prevalence of antibodies against the sialic acid 'xenoautoantigen' Neu5Gc in humans and the need for a standardised approach to quantification.

Despite an array of hypothesised implications for health, disease, and therapeutic development, antibodies against the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc) remain a subject of much debate. This systematic review of 114 publications aimed to generate a comprehensive overview of published studies in this field, addressing both the reported prevalence of anti-Neu5Gc antibodies in the human population and whether experimental variation accounts for the conflicting reports about the extent of this response. Absolute titres of anti-Neu5Gc antibodies, the reported prevalence of these antibodies, and the individual variation observed within experiments were analysed and grouped according to biological context ('inflammation', 'xenotransplantation', 'biotherapeutic use', 'cancer', and 'healthy populations'), detection method, target epitope selection, and choice of blocking agent. These analyses revealed that the experimental method had a notable impact on both the reported prevalence and absolute titres of anti-Neu5Gc antibodies in the general population, thereby limiting the ability to ascribe reported trends to genuine biological differences or the consequence of experimental design. Overall, this review highlights important knowledge gaps in the study of antibodies against this important xenoautoantigen and the need to establish a standardised method for their quantification if the extent of the importance of Neu5Gc in human health is to be fully understood.

ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) synthesis of Siglec ligands mediates anti-tumour immunity in prostate cancer.

Immune checkpoint blockade has yet to produce robust anti-cancer responses for prostate cancer. Sialyltransferases have been shown across several solid tumours, including breast, melanoma, colorectal and prostate to promote immune suppression by synthesising sialoglycans, which act as ligands for Siglec receptors. We report that ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (ST3Gal1) levels negatively correlate with androgen signalling in prostate tumours. We demonstrate that ST3Gal1 plays an important role in modulating tumour immune evasion through the synthesises of sialoglycans with the capacity to engage the Siglec-7 and Siglec-9 immunoreceptors preventing immune clearance of cancer cells. Here, we provide evidence of the expression of Siglec-7/9 ligands and their respective immunoreceptors in prostate tumours. These interactions can be modulated by enzalutamide and may maintain immune suppression in enzalutamide treated tumours. We conclude that the activity of ST3Gal1 is critical to prostate cancer anti-tumour immunity and provide rationale for the use of glyco-immune checkpoint targeting therapies in advanced prostate cancer.

The catalytic subunit of DNA-PK regulates transcription and splicing of AR in advanced prostate cancer.

Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V-positive patients with PC.

CDC20 Is Regulated by the Histone Methyltransferase, KMT5A, in Castration-Resistant Prostate Cancer.

The methyltransferase KMT5A has been proposed as an oncogene in prostate cancer and therefore represents a putative therapeutic target. To confirm this hypothesis, we have performed a microarray study on a prostate cancer cell line model of androgen independence following KMT5A knockdown in the presence of the transcriptionally active androgen receptor (AR) to understand which genes and cellular processes are regulated by KMT5A in the presence of an active AR. We observed that 301 genes were down-regulated whilst 408 were up-regulated when KMT5A expression was reduced. KEGG pathway and gene ontology analysis revealed that apoptosis and DNA damage signalling were up-regulated in response to KMT5A knockdown whilst protein folding and RNA splicing were down-regulated. Under these conditions, the top non-AR regulated gene was found to be CDC20, a key regulator of the spindle assembly checkpoint with an oncogenic role in several cancer types. Further investigation revealed that KMT5A regulates CDC20 in a methyltransferase-dependent manner to modulate histone H4K20 methylation within its promoter region and indirectly via the p53 signalling pathway. A positive correlation between KMT5A and CDC20 expression was also observed in clinical prostate cancer samples, further supporting this association. Therefore, we conclude that KMT5A is a valid therapeutic target for the treatment of prostate cancer and CDC20 could potentially be utilised as a biomarker for effective therapeutic targeting.

Engineering prostate cancer in vitro: what does it take?

A key challenge in the clinical management and cause of treatment failure of prostate cancer (PCa) is its molecular, cellular and clinical heterogeneity. Modelling systems that fully recapitulate clinical diversity and resistant phenotypes are urgently required for the development of successful personalised PCa therapies. The advent of the three-dimensional (3D) organoid model has revolutionised preclinical cancer research through reflecting heterogeneity and offering genomic and environmental manipulation that has opened up unparalleled opportunities for applications in disease modelling, high-throughput drug screening and precision medicine. Despite these remarkable achievements of organoid technology, several shortcomings in emulating the complex tumor microenvironment and dynamic process of metastasis as well as the epigenome profile limit organoids achieving true in vivo functionality. Technological advances in tissue engineering have enabled the development of innovative tools to facilitate the design of improved 3D cancer models. In this review, we highlight the current in vitro 3D PCa models with a special focus on organoids and discuss engineering approaches to create more physiologically relevant PCa organoid models and maximise their translational relevance that ultimately will help to realise the transformational power of precision medicine.

Human prostate organoid generation and the identification of prostate development drivers using inductive rodent tissues.

The reactivation of developmental genes and pathways during adulthood may contribute to pathogenesis of diseases such as prostate cancer. Analysis of the mechanistic links between development and disease could be exploited to identify signalling pathways leading to disease in the prostate. However, the mechanisms underpinning prostate development require further characterisation to interrogate fully the link between development and disease. Previously, our group developed methods to produce prostate organoids using induced pluripotent stem cells (iPSCs). Here, we show that human iPSCs can be differentiated into prostate organoids using neonatal rat seminal vesicle mesenchyme in vitro. The organoids can be used to study prostate development or modified to study prostate cancer. We also elucidated molecular drivers of prostate induction through RNA-sequencing analyses of the rat urogenital sinus and neonatal seminal vesicles. We identified candidate drivers of prostate development evident in the inductive mesenchyme and epithelium involved with prostate specification. Our top candidates included Spx, Trib3, Snai1, Snai2, Nrg2 and Lrp4. This work lays the foundations for further interrogation of the reactivation of developmental genes in adulthood, leading to prostate disease.

Traumatic arteriovenous fistula from internal maxillary artery following trigeminal balloon compression.

We describe the development of a direct traumatic arteriovenous fistula arising from the internal maxillary artery after an uneventful percutaneous trigeminal balloon compression for trigeminal neuralgia, and its management through embolization and radiosurgery.

Understanding the implementation of 'Making Every Contact Count' (MECC) delivered by healthcare professionals in a mental health hospital: protocol for a pragmatic formative process evaluation.

Background: 'Making Every Contact Count' (MECC) is a public health strategy supporting public-facing workers to use opportunities during routine contacts to enable health behaviour change. A mental health hospital in the North East of England is currently implementing a programme to embed MECC across the hospital supporting weight management ('A Weight Off Your Mind'). Bespoke MECC training has been developed to improve staff confidence in discussing physical activity, healthy eating, and related behaviour change with service users. This article describes the protocol for a pragmatic formative process evaluation to inform the implementation plan for MECC and facilitate successful implementation of the bespoke MECC training at scale. Methods/Design: An 18-month, mixed method pragmatic formative process evaluation, including qualitative research, surveys, document review and stakeholder engagement. This project is conducted within a mental health inpatient setting in the North East of England. Programme documents will be reviewed, mapped against MECC national guidelines, Behaviour Change Techniques (BCTs) and intervention functions within the Behaviour Change Wheel. A cross-sectional survey (n = 365) and qualitative semi-structured interviews (n = 30) will be conducted with healthcare practitioners delivering MECC to assess capability, opportunity and motivation. Data collection and fidelity procedures will be examined, including design, training and delivery dimensions of fidelity. Interviews with service users (n = 20) will also be conducted. Discussion: Anticipated outcomes include developing recommendations to overcome barriers to delivery of and access to MECC, including whether to either support the use of the existing MECC protocol or tailor the MECC training programme. The findings are anticipated to improve fidelity of MECC training within mental health inpatient settings as well as provide evidence for MECC training at a national level. We also expect findings to influence strategic plans, policy, and practice specific to MECC and inform best practice in implementing wider brief intervention programmes.

A Review of Prostate Organogenesis and a Role for iPSC-Derived Prostate Organoids to Study Prostate Development and Disease.

The prostate is vulnerable to two major age-associated diseases, cancer and benign enlargement, which account for significant morbidity and mortality for men across the globe. Prostate cancer is the most common cancer reported in men, with over 1.2 million new cases diagnosed and 350,000 deaths recorded annually worldwide. Benign prostatic hyperplasia (BPH), characterised by the continuous enlargement of the adult prostate, symptomatically afflicts around 50% of men worldwide. A better understanding of the biological processes underpinning these diseases is needed to generate new treatment approaches. Developmental studies of the prostate have shed some light on the processes essential for prostate organogenesis, with many of these up- or downregulated genes expressions also observed in prostate cancer and/or BPH progression. These insights into human disease have been inferred through comparative biological studies relying primarily on rodent models. However, directly observing mechanisms of human prostate development has been more challenging due to limitations in accessing human foetal material. Induced pluripotent stem cells (iPSCs) could provide a suitable alternative as they can mimic embryonic cells, and iPSC-derived prostate organoids present a significant opportunity to study early human prostate developmental processes. In this review, we discuss the current understanding of prostate development and its relevance to prostate-associated diseases. Additionally, we detail the potential of iPSC-derived prostate organoids for studying human prostate development and disease.

Co-localisation of intra-nuclear membrane type-1 matrix metalloproteinase and hypoxia inducible factor-2α in osteosarcoma and prostate carcinoma cells.

Increased membrane type-1 matrix metalloproteinase (MT1-MMP) expression in osteosarcoma is predictive of poor prognosis and directs bone metastasis in prostate carcinoma. MT1-MMP subcellular localisation varies with oxygen tension, and, therefore, the aim of the present study was to assess protein interactions between MT1-MMP and the hypoxia inducible factors (HIF-1α and HIF-2α). MT1-MMP protein expression was investigated across a panel of cancer cell lines, including a positive and negative control. The hypoxia-induced alteration in subcellular location of MT1-MMP, HIF-1α and HIF-2α in the U2OS osteosarcoma cell line was assessed using subcellular fractionation. A proximity ligation assay was utilised to assess protein to protein interactions in the osteosarcoma U2OS and prostate carcinoma PC3 cell lines. U2OS and PC3 cells exhibited a significantly increased intra-nuclear interaction between MT1-MMP and HIF-2α in response to hypoxia. The role of this warrants further investigation as it may unveil novel opportunities to target MT1-MMP, which is of particular significance for osteosarcoma since current treatment options are limited.

Towards an End-to-End Framework of CCTV-Based Urban Traffic Volume Detection and Prediction.

Near real-time urban traffic analysis and prediction are paramount for effective intelligent transport systems. Whilst there is a plethora of research on advanced approaches to study traffic recently, only one-third of them has focused on urban arterials. A ready-to-use framework to support decision making in local traffic bureaus using largely available IoT sensors, especially CCTV, is yet to be developed. This study presents an end-to-end urban traffic volume detection and prediction framework using CCTV image series. The framework incorporates a novel Faster R-CNN to generate vehicle counts and quantify traffic conditions. Then it investigates the performance of a statistical-based model (SARIMAX), a machine learning (random forest; RF) and a deep learning (LSTM) model to predict traffic volume 30 min in the future. Tests at six locations with varying traffic conditions under different lengths of past time series are used to train the prediction models. RF and LSTM provided the most accurate predictions, with RF being faster than LSTM. The developed framework has been successfully applied to fill data gaps under adverse weather conditions when data are missing. It can be potentially implemented in near real time at any CCTV location and integrated into an online visualization platform.

Same day embolisation followed by microsurgical resection of brain arteriovenous malformations: a single centre early experience.

INTRODUCTION: The purpose of this paper is to report our experience of treating cerebral arteriovenous malformations (AVM) in adults with pre-operative embolisation and microsurgical resection on the same day during a single anaesthetic at a single centre between April 2016 and December 2018. We included both elective AVM and AVM that had bled acutely. METHODS: We retrospectively analysed data from patients with cerebral AVMs who underwent embolisation followed by microsurgical resection on the same day at a single neurosurgical centre. PRIMARY ENDPOINTS INCLUDED: Total procedure time (embolisation and microsurgical resection), procedure finish time, intra-operative blood loss, degree of nidus obliteration on postoperative angiography, intensive care unit (ICU) stay, total stay at the neurosurgical centre and modified Rankin Score (pre- and post-procedure). RESULTS: •Nineteen patients underwent same-day pre-operative embolisation and microsurgical resection over the 32-month period. The average patient age was 40 years (range 19-66 years). One patient had undergone a prior attempt at embolisation and one patient previously had sterotactic radiosurgery (STRS). •Thirteen of the AVM were in the dominant hemisphere and six in the non-dominant hemisphere. Sixteen AVM were located supratentorially and three were in the posterior fossa. Spetzler-Martin grades included 4 grade 1, 10 grade 2, 4 grade 3 and 1 grade 4. •The average blood loss intra-operatively was 289 mls. •The average list finish time was 19:56 (range 15:10-00:00). •Seventeen patients had 100% nidus obliteration on post-operative digital subtraction angiography, one patient had a small remnant and was referred to STRS and one patient died in the ICU post operatively. CONCLUSION: Overall, the authors believe same-day embolisation and microsurgical resection represents a safe treatment strategy. The technique minimises hemorrhagic complications from delayed venous occlusion and avoids multiple anaesthetics and hospital admissions.

IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway.

Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.

Propagation of human prostate tissue from induced pluripotent stem cells.

Primary culture of human prostate organoids and patient-derived xenografts is inefficient and has limited access to clinical tissues. This hampers their use for translational study to identify new treatments. To overcome this, we established a complementary approach where rapidly proliferating and easily handled induced pluripotent stem cells enabled the generation of human prostate tissue in vivo and in vitro. By using a coculture technique with inductive urogenital sinus mesenchyme, we comprehensively recapitulated in situ 3D prostate histology, and overcame limitations in the primary culture of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. This model now unlocks new opportunities to undertake translational studies of benign and malignant prostate disease.

Correction: BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (PtenΔ/Δ BRF1Tg) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In PtenΔ/Δ BRF1Tg tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis.

A novel CRISPR-engineered prostate cancer cell line defines the AR-V transcriptome and identifies PARP inhibitor sensitivities.

Resistance to androgen receptor (AR)-targeted therapies in prostate cancer (PC) is a major clinical problem. A key mechanism of treatment resistance in advanced PC is the generation of alternatively spliced forms of the AR termed AR variants (AR-Vs) that are refractory to targeted agents and drive tumour progression. Our understanding of how AR-Vs function is limited due to difficulties in distinguishing their discriminate activities from full-length AR (FL-AR). Here we report the development of a novel CRISPR-derived cell line which is a derivative of CWR22Rv1 cells, called CWR22Rv1-AR-EK, that has lost expression of FL-AR, but retains all endogenous AR-Vs. From this, we show that AR-Vs act unhindered by loss of FL-AR to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrate that AR-Vs drive expression of a cohort of DNA damage response genes and depletion of AR-Vs sensitises cells to ionising radiation. Moreover, we demonstrate that AR-Vs interact with PARP1 and PARP2 and are dependent upon their catalytic function for transcriptional activation. Importantly, PARP blockade compromises expression of AR-V-target genes and reduces growth of CRPC cell lines suggesting a synthetic lethality relationship between AR-Vs and PARP, advocating the use of PARP inhibitors in AR-V positive PC.

The Pioneering Role of GATA2 in Androgen Receptor Variant Regulation Is Controlled by Bromodomain and Extraterminal Proteins in Castrate-Resistant Prostate Cancer.

The androgen receptor (AR) is a key driver of prostate cancer development. Antiandrogens effectively inactivate the AR, but subsequent AR reactivation progresses the disease to castrate-resistant prostate cancer (CRPC). Constitutively active AR splice variants (AR-V) that function unchallenged by current AR-targeted therapies are key drivers of CRPC. Currently, very little is known about the regulation of AR-Vs at the chromatin level. Here, we show that the pioneer factor GATA2 is a critical regulator of AR-Vs. Furthermore, we demonstrate that the GATA2 cistrome in CRPC shares considerable overlap with bromodomain and extraterminal (BET) proteins and is codependent for DNA binding. GATA2 activity is compromised by BET inhibitors, which attenuates the pioneering role of GATA2 in CRPC. In all, this study indicates that GATA2 is a critical regulator of AR-V-mediated transactivation and is sensitive to BET inhibitors, signifying these agents may be efficacious in patients with CRPC which overexpress GATA2. IMPLICATIONS: We have defined novel mechanisms of AR-V and GATA2 regulation in advanced prostate cancer that could be therapeutically exploited.