Glutamine deprivation alters the origin and function of cancer cell exosomes.

Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies (MVBs). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTPase Rab11, generated in Rab11-positive recycling endosomal MVBs. Release of Rab11-positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 (mTORC1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo. Their growth-promoting activity, which is also observed in vitro, is Rab11a-dependent, involves ERK-MAPK-signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC1 inhibition stimulates release from Rab11a compartments of exosomes with pro-tumorigenic functions, which we propose promote stress-induced tumour adaptation.

Remote monitoring of patients with rheumatoid arthritis in a low disease activity state: a mixed methods evaluation across six hospitals in London, UK.

OBJECTIVES: This study evaluated the scale-up of a remote monitoring service, capturing monthly Rheumatoid Arthritis Impact of Disease scores and patient-generated text messages, for patients with rheumatoid arthritis (RA; in remission or with low disease activity) attending routine outpatient clinics across six hospitals. We explored patients and staff experiences and implementation outcomes. METHODS: A pragmatic, mixed methods approach was used, with active patient involvement throughout. We undertook a rapid review, analysed service-level data, and conducted a patient survey and patient and staff interviews, informed by the Capability, Opportunity, Motivation, Behaviour (COM-B) and Exploration, Preparation, Implementation, Sustainment (EPIS) theoretical frameworks. RESULTS: The review included 37 articles, covering themes of patient and clinician acceptability, engagement, feasibility and clinical impact. Service-level data (n = 202) showed high levels of patient engagement with the service. The patient survey (n = 155) showed patients felt the service was easy to use, had confidence in it and felt it improved access to care. Patient interview (n = 22) findings mirrored those of the survey. Motivating factors included increased responsiveness and ease of contact with clinical teams. Views from staff interviews (n = 16) were more mixed. Some implementation barriers were specific to roll-out sites. Prioritization of staff needs was emphasized. CONCLUSION: Patients were positive about the service and engagement was high. Staff views and engagement were more mixed. Results suggest that equal levels of patient and staff engagement are required for sustainability. These findings further our understanding of the implementation challenges to scaling remote monitoring interventions for patients with rheumatoid arthritis in routine care settings.

Single cell RNA-sequencing: A powerful yet still challenging technology to study cellular heterogeneity.

Almost all biomedical research to date has relied upon mean measurements from cell populations, however it is well established that what it is observed at this macroscopic level can be the result of many interactions of several different single cells. Thus, the observable macroscopic 'average' cannot outright be used as representative of the 'average cell'. Rather, it is the resulting emerging behaviour of the actions and interactions of many different cells. Single-cell RNA sequencing (scRNA-Seq) enables the comparison of the transcriptomes of individual cells. This provides high-resolution maps of the dynamic cellular programmes allowing us to answer fundamental biological questions on their function and evolution. It also allows to address medical questions such as the role of rare cell populations contributing to disease progression and therapeutic resistance. Furthermore, it provides an understanding of context-specific dependencies, namely the behaviour and function that a cell has in a specific context, which can be crucial to understand some complex diseases, such as diabetes, cardiovascular disease and cancer. Here, we provide an overview of scRNA-Seq, including a comparative review of emerging technologies and computational pipelines. We discuss the current and emerging applications and focus on tumour heterogeneity a clear example of how scRNA-Seq can provide new understanding of a complex disease. Additionally, we review the limitations and highlight the need of powerful computational pipelines and reproducible protocols for the broader acceptance of this technique in basic and clinical research.

Hypoxia-induced switch in SNAT2/SLC38A2 regulation generates endocrine resistance in breast cancer.

Tumor hypoxia is associated with poor patient outcomes in estrogen receptor-α-positive (ERα+) breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here, we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer, and is regulated by hypoxia both in vitro and in vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ERα, but it became predominantly a hypoxia-inducible factor 1α (HIF-1α)-dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1α and ERα overlap in SNAT2's cis-regulatory elements. In addition, the down-regulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia. Overexpression of SNAT2 in vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ERα inhibition, in hypoxia, or when glutamine levels were low. SNAT2 up-regulation in vivo caused complete resistance to antiestrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlated with hypoxia profiles and worse outcome in patients given antiestrogen therapies. Our findings show a switch in the regulation of SNAT2 between ERα and HIF-1α, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer.

ELTD1 Activation Induces an Endothelial-EMT Transition to a Myofibroblast Phenotype.

ELTD1 is expressed in endothelial and vascular smooth muscle cells and has a role in angiogenesis. It has been classified as an adhesion GPCR, but as yet, no ligand has been identified and its function remains unknown. To establish its role, ELTD1 was overexpressed in endothelial cells. Expression and consequently ligand independent activation of ELTD1 results in endothelial-mesenchymal transistion (EndMT) with a loss of cell-cell contact, formation of stress fibres and mature focal adhesions and an increased expression of smooth muscle actin. The effect was pro-angiogenic, increasing Matrigel network formation and endothelial sprouting. RNA-Seq analysis after the cells had undergone EndMT revealed large increases in chemokines and cytokines involved in regulating immune response. Gene set enrichment analysis of the data identified a number of pathways involved in myofibroblast biology suggesting that the endothelial cells had undergone a type II EMT. This type of EMT is involved in wound repair and is closely associated with inflammation implicating ELTD1 in these processes.

RHOQ is induced by DLL4 and regulates angiogenesis by determining the intracellular route of the Notch intracellular domain.

Angiogenesis, the formation of new blood vessels by endothelial cells, is a finely tuned process relying on the balance between promoting and repressing signalling pathways. Among these, Notch signalling is critical in ensuring appropriate response of endothelial cells to pro-angiogenic stimuli. However, the downstream targets and pathways effected by Delta-like 4 (DLL4)/Notch signalling and their subsequent contribution to angiogenesis are not fully understood. We found that the Rho GTPase, RHOQ, is induced by DLL4 signalling and that silencing RHOQ results in abnormal sprouting and blood vessel formation both in vitro and in vivo. Loss of RHOQ greatly decreased the level of Notch signalling, conversely overexpression of RHOQ promoted Notch signalling. We describe a new feed-forward mechanism regulating DLL4/Notch signalling, whereby RHOQ is induced by DLL4/Notch and is essential for the NICD nuclear translocation. In the absence of RHOQ, Notch1 becomes targeted for degradation in the autophagy pathway and NICD is sequestered from the nucleus and targeted for degradation in lysosomes.

A small molecule targeting ALK1 prevents Notch cooperativity and inhibits functional angiogenesis.

Activin receptor-like kinase 1 (ALK1, encoded by the gene ACVRL1) is a type I BMP/TGF-ß receptor that mediates signalling in endothelial cells via phosphorylation of SMAD1/5/8. During angiogenesis, sprouting endothelial cells specialise into tip cells and stalk cells. ALK1 synergises with Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2 and thereby represses tip cell formation and angiogenic sprouting. The ALK1-Fc soluble protein fusion has entered clinic trials as a therapeutic strategy to sequester the high-affinity extracellular ligand BMP9. Here, we determined the crystal structure of the ALK1 intracellular kinase domain and explored the effects of a small molecule kinase inhibitor K02288 on angiogenesis. K02288 inhibited BMP9-induced phosphorylation of SMAD1/5/8 in human umbilical vein endothelial cells to reduce both the SMAD and the Notch-dependent transcriptional responses. In endothelial sprouting assays, K02288 treatment induced a hypersprouting phenotype reminiscent of Notch inhibition. Furthermore, K02288 caused dysfunctional vessel formation in a chick chorioallantoic membrane assay of angiogenesis. Such activity may be advantageous for small molecule inhibitors currently in preclinical development for specific BMP gain of function conditions, including diffuse intrinsic pontine glioma and fibrodysplasia ossificans progressiva, as well as more generally for other applications in tumour biology.

The role of the dementia specialist nurse in acute care: a scoping review.

AIMS AND OBJECTIVES: To identify the potential benefits of dementia specialist nursing and to inform the implementation of roles to support people with dementia during hospital admission. BACKGROUND: Extended stays and adverse events mean that hospital admissions are costly for people with dementia, and patient experiences and outcomes can be poor. Specialist nurses have been identified as having potential to enhance care quality, reduce excess stays and reduce costs, but the evidence base for dementia specialist nurse roles has not previously been synthesised. DESIGN: Scoping review. DATA SOURCES: Cochrane Library, Campbell Collaboration, Clinical Evidence, Evidence-Based Medicine, York Centre for Reviews and Dissemination, PubMed, Medline, CINAHL and PsycInfo databases and internet searches and personal libraries/expert consultation to identify grey literature. METHODS: Initial scoping searches were used to inform more focused systematic searches. Studies directly evaluating dementia nurse specialist roles or giving evidence of effectiveness of interventions/services that could be delivered by them to improve core outcomes were identified by one reviewer and verified by a second reviewer. RESULTS: While direct evidence for the effectiveness of these roles is lacking, a number of areas were identified in which a nurse specialist role could make a contribution, including preventing adverse events and improving patient experiences and outcomes. There is a considerable body of evidence for the effectiveness of these interventions although the volume of evidence for specific interventions is not always significant. CONCLUSIONS: The evidence indicates that a skilled dementia specialist nurse, undertaking a clearly defined role, and working directly with people with dementia and their carers for a significant proportion of the time, could benefit people with dementia in hospitals and their family carers. RELEVANCE TO CLINICAL PRACTICE: Clear guidance for the development and implementation of dementia specialist nurse roles in acute hospital settings.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes.

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

Providing easier access to community-based healthcare for people with joint pain: Experiences of delivering ESCAPE-pain in community venues by exercise professionals.

BACKGROUND: Joint pain adversely impacts the physical, mental, socioeconomic and emotional wellbeing of many millions of people. Enabling Self-management and Coping with Arthritic Pain using Exercise, ESCAPE-pain, is a rehabilitation programme that reduces joint pain and its impact. The programme is usually delivered in clinical settings by physiotherapists but delivering it in community venues would improve access greatly. AIM: To explore the feasibility of delivering ESCAPE-pain in community venues, and the experiences of organisations and facilitators delivering it. METHODS: Semi-structured interviews were conducted with managers of 17 community organisations and 10 facilitators. RESULTS: People were happy to attend ESCAPE-pain delivered by exercise professionals at community venues, which they found convenient and valuable. It expanded community organisation's offer to older people, utilised their facilities off-peak and advanced facilitator's personal and professional development. Recruitment onto the programme was easiest where there were good links with local clinical providers. Although collecting outcome data was burdensome it demonstrated the programme's effectiveness to commissioners. Some clinical commissioners contracted community organisations to deliver ESCAPE-pain reducing their costs and freeing up clinical facilities. Organisations also financed ESCAPE-pain by charging participants a nominal fee for the programme, post-programme classes to support participants remain active and/or a membership fee. CONCLUSIONS: ESCAPE-pain delivered in community venues facilitated access to better care and on-going support. Partnerships between healthcare commissioners and community providers maximised efficient use of their facilities and resources and fulfilled national policy of encouraging self-management of long-term conditions in the community.

ELTD1 is present in extracellular vesicles derived from endothelial cells as a cleaved extracellular domain which induces in vivo angiogenesis.

ELTD1/ADGRL4 is an adhesion GPCR with an important role in angiogenesis. We recently identified a role for ELTD1 in wound repair and inflammation. Activation of ELTD1 in endothelial cells results in a type II EMT to myofibroblast-like cells that have enhanced angiogenic ability. Furthermore, expression of Eltd1 in murine breast cancer cells increases tumour growth by increasing blood vessel size and perfusion and by creating an immunosuppressive microenvironment. As extracellular vesicles (EVs) are known to be involved in vascular development, growth and maturation we investigated the composition and functional effects of the EVs isolated from ELTD1 expressing cells to elucidate their role in these processes. A highly glycosylated form of the extracellular domain (ECD) of ELTD1 is readily incorporated into EVs. Using mass spectrometry-based proteomics we identified proteins that are enriched in ELTD1-EVs and are involved in haemostasis and immune responses. ELTD1 enriched EVs were pro-angiogenic in vivo and in vitro and the presence of the ECD alone induced endothelial sprouting. In endothelial cells experiencing laminar flow, ELTD1 levels were reduced in the EVs when they are quiescent, showing a relationship between ELTD1 and the activation state of the endothelium. Using FACS, we detected a significant increase in vesicular ELTD1 in the plasma of patients with preeclampsia, a condition characterized by endothelial dysfunction. These data confirm a role for ELTD1 in wound repair and inflammation and reveal its potential as a biomarker of vessel dysfunction.

Investigations on Zinc Isotope Fractionation in Breast Cancer Tissue Using in vitro Cell Culture Uptake-Efflux Experiments.

Zinc (Zn) accumulates in breast cancer tumors compared to adjacent healthy tissue. Clinical samples of breast cancer tissue show light Zn isotopic compositions (δ66Zn) relative to healthy tissue. The underlying mechanisms causing such effects are unknown. To investigate if the isotopic discrimination observed for in vivo breast cancer tissue samples can be reproduced in vitro, we report isotopic data for Zn uptake-efflux experiments using a human breast cancer cell line. MDA-MB-231 cell line was used as a model for triple receptor negative breast cancer. We determined Zn isotope fractionation for Zn cell uptake (Δ66Znuptake) and cell efflux (Δ66Znefflux) using a drip-flow reactor to enable comparison with the in vivo environment. The MDA-MB-231 cell line analyses show Zn isotopic fractionations in an opposite direction to those observed for in vivo breast cancer tissue. Uptake of isotopically heavy Zn (Δ66Znuptake = +0.23 ± 0.05‰) is consistent with transport via Zn transporters (ZIPs), which have histidine-rich binding sites. Zinc excreted during efflux is isotopically lighter than Zn taken up by the cells (Δ66Znefflux = -0.35 ± 0.06‰). The difference in Zn isotope fractionation observed between in vitro MDA-MB-231 cell line experiments and in vivo breast tissues might be due to differences in Zn transporter levels or intercellular Zn storage (endoplasmic reticulum and/or Zn specific vesicles); stromal cells, such as fibroblasts and immune cells. Although, additional experiments using other human breast cancer cell lines (e.g., MCF-7, BT-20) with varying Zn protein characteristics are required, the results highlight differences between in vitro and in vivo Zn isotope fractionation.

Elevated expression of the adhesion GPCR ADGRL4/ELTD1 promotes endothelial sprouting angiogenesis without activating canonical GPCR signalling.

ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways.

ADGRL4/ELTD1 Expression in Breast Cancer Cells Induces Vascular Normalization and Immune Suppression.

ELTD1/ADGRL4 expression is increased in the vasculature of a number of tumor types and this correlates with a good prognosis. Expression has also been reported in some tumor cells with high expression correlating with a good prognosis in hepatocellular carcinoma (HCC) and a poor prognosis in glioblastoma. Here we show that 35% of primary human breast tumors stain positively for ELTD1, with 9% having high expression that correlates with improved relapse-free survival. Using immunocompetent, syngeneic mouse breast cancer models we found that tumors expressing recombinant murine Eltd1 grew faster than controls, with an enhanced ability to metastasize and promote systemic immune effects. The Eltd1-expressing tumors had larger and better perfused vessels and tumor-endothelial cell interaction led to the release of proangiogenic and immune-modulating factors. M2-like macrophages increased in the stroma along with expression of programmed death-ligand 1 (PD-L1) on tumor and immune cells, to create an immunosuppressive microenvironment that allowed Eltd1-regulated tumor growth in the presence of an NY-ESO-1-specific immune response. Eltd1-positive tumors also responded better to chemotherapy which could explain the relationship to a good prognosis observed in primary human cases. Thus, ELTD1 expression may enhance delivery of therapeutic antibodies to reverse the immunosuppression and increase response to chemotherapy and radiotherapy in this subset of tumors. ELTD1 may be useful as a selection marker for such therapies. IMPLICATIONS: ELTD1 expression in mouse breast tumors creates an immunosuppressive microenvironment and increases vessel size and perfusion. Its expression may enhance the delivery of therapies targeting the immune system.

Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors.

This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2-hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4-green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2-hybrid and glutathione-S-transferase pulldown analyses show Robo4 binding to a Wiskott-Aldrich syndrome protein (WASP), neural Wiskott-Aldrich syndrome protein, and WASP-interacting protein actin-nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4-mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation-promoting factors to promote endothelial cell migration.

Functionally defining the endothelial transcriptome, from Robo4 to ECSCR.

We have applied search algorithms to expression databases to identify genes whose expression is restricted to the endothelial cell. Such genes frequently play a critical role in endothelial biology and angiogenesis. Two such genes are the roundabout receptor Robo4 and the ECSCR (endothelial-cell-specific chemotaxis regulator). Endothelial cells express both Robo1 and Robo4, which we have knocked down using siRNA (small interfering RNA) and then studied the effect in a variety of in vitro assays. Both Robo4 and Robo1 knockdown inhibited in vitro tube formation on Matrigel. Transfection of Robo4 into endothelial cells increased the number of filopodial extensions from the cell, but failed to do so in Robo1-knockdown cells. Separate immunoprecipitation studies showed that Robo1 and Robo4 heterodimerize. We conclude from this and other work that a heteroduplex of Robo1 and Robo4 signals through WASP (Wiskott-Aldrich syndrome protein) and other actin nucleation-promoting factors to increase the number of filopodia and cell migration. Knockdown of the transmembrane ECSCR protein in endothelial cells also reduced chemotaxis and impaired tube formation on Matrigel. Yeast two-hybrid analysis and immunoprecipitation studies showed that, in contrast with the roundabouts, ECSCR binds to the actin-modulatory filamin A. We conclude that all three of these genes are critical for effective endothelial cell migration and, in turn, angiogenesis.

Copper chelator ATN-224 inhibits endothelial function by multiple mechanisms.

Copper is required for the proliferation of endothelial cells and copper-lowering therapy reduces tumour growth in animal models. It has been reported that ATN-224, a novel copper chelator, potently inhibits the activity of the copper-dependent enzyme superoxide dismutase 1 (SOD1) in endothelial cells. We performed microarray analysis of gene expression in endothelial cells exposed to ATN-224 which revealed upregulation of stress response genes including heme-oxygenase 1 (HO-1) and differential regulation of several genes previously implicated in angiogenesis including CXCR4, ANGP2, PGES2, RHAMM, ITB4 and AQP1 (p<0.01). These changes were confirmed on qPCR. Treatment of HUVEC with ATN-224 caused increased superoxide levels, phospho-ERK signalling, nuclear NRF1 expression, HO-1 expression and induction of the anti-apoptotic proteins P21, BCL2 and BCLXL. There was also nuclear translocation of SOD1. SOD1 RNA interference replicated the effects of ATN-224 on endothelial cell function but did not cause upregulation of HO-1 or PGES2, suggesting additional mechanisms of action of ATN-224. Downregulation of AQP1, which has been shown to have a role in angiogenesis, was seen with both ATN-224 and SOD1 siRNA. AQP1 expression could be rescued after ATN-224 by added copper. RNA interference to AQP1 inhibited endothelial proliferation and migration, confirming the role of AQP1 in endothelial cell function. Therefore regulation of AQP1 may represent an important action of copper chelation therapy.

hsa-miR-210 Is induced by hypoxia and is an independent prognostic factor in breast cancer.

PURPOSE: MicroRNA (miRNA) expression alterations have been described in cancer. Many cancers are characterized by areas of hypoxia, enhanced hypoxia-inducible factor (HIF) levels, and increased expression of hypoxically regulated genes, all of which correlate with patient outcome. We examined hypoxia-induced miRNA expression changes to identify markers of survival in breast cancer. EXPERIMENTAL DESIGN: We used microarrays to analyze miRNA expression changes induced by hypoxia in MCF7 breast cancer cell lines and validated results by quantitative-PCR (Q-PCR). Small interfering RNA against HIF-1alpha and HIF-2alpha, and RCC4 cells transfected with the von Hippel-Lindau (VHL) protein were used to investigate HIF dependency of miRNA expression. miRNA Q-PCR assays were done on 219 early breast cancer samples with long-term follow-up. Correlation of expression with clinical variables was done using Pearson and Spearman's rank tests, univariate, and Cox multivariate analysis. RESULTS: hsa-miR-210 induction was the most significant change under hypoxia by microarray analysis (3.4-fold, P < 0.001). hsa-miR-210 expression changes were validated by Q-PCR and detected in other cancer cell lines. Using small interfering RNAs and RCC4 cells transfected with VHL, we showed that the regulation by hypoxia of hsa-miR-210 was mediated by the HIF-1alpha/VHL transcriptional system but not HIF-2alpha. hsa-miR-210 expression levels in breast cancer samples correlated directly with a hypoxia score based on the expression of 99 genes. hsa-miR-210 expression levels showed an inverse correlation with disease-free and overall survival, significant in both univariate and multivariate analyses. CONCLUSIONS: We show that hsa-miR-210 overexpression is induced by hypoxia in a HIF-1alpha- and VHL-dependent fashion and its expression levels in breast cancer samples are an independent prognostic factor.

ADGRL4/ELTD1 Silencing in Endothelial Cells Induces ACLY and SLC25A1 and Alters the Cellular Metabolic Profile.

Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1's full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1's role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of ACLY (fatty acid and cholesterol synthesis or acetylation). Silencing induced KIT expression and affected the Notch signalling pathway, upregulating Delta Like Canonical Notch Ligand 4 (DLL4) and suppressing Jagged Canonical Notch Ligand 1 (JAG1) and Hes Family BHLH Transcription Factor 2 (HES2). The effect of ADGRL4/ELTD1 silencing on the cellular metabolic profile was modest but several metabolites were significantly affected. Cis-aconitic acid, uridine diphosphate (UDP)-glucoronate, fructose 2,6-diphosphate, uridine 5-diphosphate, and aspartic acid were all elevated as a result of silencing and phosphocreatine, N-acetylglutamic acid, taurine, deoxyadenosine triphosphate, and cytidine monophosphate were depleted. Metabolic pathway analysis implicated ADGRL4/ELTD1 in pyrimidine, amino acid, and sugar metabolism. In summary, this study shows that ADGRL4/ELTD1 impacts core components of endothelial metabolism and regulates genes involved in endothelial differentiation/homeostasis and Notch signalling.