Examination of potential mechanisms linking AMPK to inhibition of IL-6 signalling.

Considerable recent evidence supports the role of AMP-activated protein kinase (AMPK) as an anti-inflammatory mediator, yet the mechanisms of its anti-inflammatory actions are only starting to be unravelled. Inappropriate cytokine stimulated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signalling is a key feature of many pro-inflammatory events, including atherogenesis. Previous unpublished studies in our group have investigated whether AMPK modifies cytokine stimulation of JAK-STAT signalling in HUVECs. These preliminary investigations demonstrated that pre-treatment of HUVECs with AMPK activator, A769662, significantly inhibits both sIL-6Rα/IL-6 and IFN-α stimulation of STAT3 Tyr705 phosphorylation in HUVECs. IFN-α activates STATs via an IFNα/β receptor 1 (IFNAR1/IFNAR2) complex which is distinct from the sIL-6Rα/IL-6/gp130 complex. The studies in this thesis therefore tested the hypothesis that AMPK was exerting its inhibitory effects at one or more common signalling loci downstream of IFNAR1/IFNAR2 and gp130 at a post-receptor level. First, it was investigated whether AMPK exerts its inhibitory effects on JAK-STAT signalling via a known regulator of JAK or STAT, or an AMPK downstream target known to either directly or indirectly impact on JAK-STAT signalling. A combination of genetic and pharmacological approaches was utilised to assess the role of each of the following AMPK targets: TC-PTP, SHP2, eNOS, PKCλ, SIRT1, CPT1 and mTOR. It was demonstrated that activation of AMPK in HUVECs inhibited sIL-6Rα/IL-6 stimulated STAT3 Tyr705 phosphorylation via a mechanism independent of TC-PTP, eNOS, PKC, SIRT1 and mTOR. Furthermore, inhibition of mTOR and eNOS reduced sIL-6Rα/IL-6 stimulated STAT3 Tyr705 phosphorylation, independent of AMPK activation by A769662. Next, it was investigated whether AMPK acts directly on a signalling component of the JAK-STAT pathway. Specifically, it was hypothesised that AMPK could directly phosphorylate serine or threonine residues within JAK to inhibit IL-6 signalling. siRNA-mediated downregulation of JAK isoforms demonstrated that IL-6 induced STAT3 Tyr705 phosphorylation predominantly via JAK1 in human umbilical vein endothelial cells (HUVECs). In vitro kinase assays of JAK1-derived peptides demonstrated that AMPK can directly phosphorylate two residues, Ser515 and Ser518, within the JAK1 SH2 domain. Subsequently, a GST- 14-3-3 pull down assay of cell lysates produced from A769662 treated JAK1- defcient U4C cells transiently expressing either wild type or S515A/S518A double mutant JAK1 demonstrated that pharmacological activation of AMPK promotes 14-3-3 binding of JAK1 via a mechanism requiring Ser515 and Ser518. Furthermore, mutation of Ser515 and Ser518 abolishes the ability of AMPK to inhibit JAK-STAT signalling by an IL-6 trans-signalling complex and from a constitutively active Val658Phe-mutated JAK1. In this study it is proposed that AMPK phosphorylation of JAK1 at Ser515 and Ser518 inhibits IL-6 stimulated JAK1 phosphorylating STAT3 by interfering with the ability of JAK1 to interact and phosphorylate the GP130 receptor and /or STAT3 and STAT1. Therefore, AMPK phosphorylation of JAK1 could potentially be a novel regulatory mechanism that could be developed as a therapy for treating chronic inflammatory diseases such as atherosclerosis

Phosphorylation of Janus kinase 1 (JAK1) by AMP-activated protein kinase (AMPK) links energy sensing to anti-inflammatory signaling

Adenosine 5′-monophosphate-activated protein kinase (AMPK) is a pivotal regulator of metabolism at cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the Src homology 2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect that required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPK-mediated inhibition of JAK-STAT signaling stimulated by either the sIL-6Ra/IL-6 complex or the expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore, our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway. 2016</p

A pilot study evaluating the use of ABCD2 score in pre-hospital assessment of patients with suspected transient ischaemic attack: experience and lessons learned

Background: Suspected transient ischaemic attack (TIA) is a common presentation to emergency medical services (EMS) in the United Kingdom (UK). Several EMS systems have adopted the ABCD2 score to aid pre-hospital risk stratification and decision-making on patient disposition, such as direct referral to an Emergency Department or specialist TIA clinic. However, the ABCD2 score, developed for hospital use, has not been validated for use in the pre-hospital context of EMS care. Methods: We conducted a pilot study to assess eligibility criteria, recruitment rates, protocol compliance, consent and follow-up procedures to inform the development of a definitive study to validate the ABCD2 tool in pre-hospital evaluation of patients with suspected TIA. Results: From 1st May–1st September 2013, nine patients with an EMS suspected diagnosis of TIA had the TIA diagnosis later confirmed by a specialist from five participating sites. This recruitment rate is comparable to stroke trials in the EMS setting. Bureaucratic obstacles and duplication of approval processes across participating sites took 13 months to resolve before recruitment commenced. Due to the initial difficulty in recruitment, a substantial amendment was approved to modify inclusion criteria, allowing patients with atrial fibrillation and/or taking anticoagulant therapy to participate in the study. Conclusions: It is possible to identify, recruit and follow up patients with suspected TIA in the EMS setting. Training large numbers of EMS staff is required as exposure to TIA patients is infrequent. Significant insight was gained into the complexity of NHS research governance mechanisms in the UK. This knowledge will facilitate the planning of a future adequately powered study to validate the ABCD2 tool in a pre-hospital setting

Genicular artery embolisation in patients with osteoarthritis of the knee (GENESIS 2): protocol for a double-blind randomised sham-controlled trial

Knee osteoarthritis is a leading cause of chronic disability and economic burden. In many patients who are not surgical candidates, existing treatment options are insufficient. Clinical evidence for a new treatment approach, genicular artery embolisation (GAE), is currently limited to single arm cohort, or small population randomised studies. This trial will investigate the use of a permanent embolic agent for embolisation of abnormal genicular arterial vasculature to reduce pain in patients with mild to moderate knee osteoarthritis. Up to 110 participants, 45 years or older, with knee pain for ≥ 3 months resistant to conservative treatment will be randomised (1:1) to GAE or a sham procedure. The treatment group will receive embolisation using 100-micron Embozene™ microspheres (Varian, a Siemens Healthineers Company) (investigational use for this indication in the UK), and the sham group will receive 0.9% saline in an otherwise identical procedure. Patients will be followed for 24 months. At 6 months, sham participants will be offered crossover to GAE. The primary endpoint is change of 4 Knee Injury and OA Outcome Score subscales (KOOS ) at 6 months post-randomisation. The study will also evaluate quality of life, health economics, imaging findings, and psychosocial pain outcomes. The primary manuscript will be submitted for publication after all participants complete 6 months of follow-up. The trial is expected to run for 3.5 years

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis

Background MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR–92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR–92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR–92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells. Methodology/Principal Findings We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR–92 expression by qRT-PCR. Expression of ERβ1, a direct miR–92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR–92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR– 92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR–92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR–92 levels in NFs but not CAFs enhanced invasion of both MCF–7 and MDA-MB–231 breast cancer epithelial cells. Conclusions miR–92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR–92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR–92 target in breast cancer

An Evaluation of Matrix-Containing and Humanised Matrix-Free 3-Dimensional Cell Culture Systems for Studying Breast Cancer.

BACKGROUND: 3D cell cultures are emerging as more physiologically meaningful alternatives to monolayer cultures for many biological applications. They are attractive because they more closely mimic in vivo morphology, especially when co-cultured with stromal fibroblasts. METHODOLOGY/PRINCIPAL FINDINGS: We compared the efficacy of 3 different 3D cell culture systems; collagen I, low attachment culture vessels and a modification of Fibrolife®, a specialised humanised cell culture medium devoid of animal-derived components, using breast cancer cell lines representative of the different molecular subtypes of breast cancer, cultured alone or with human mammary fibroblasts with a view to developing matrix-free humanised systems. 3D collagen I culture supported the growth of a range of breast cancer cell lines. By modifying the composition of Fibrolife® to epiFL, matrix-free cell culture was possible. During sequential transfer to epiFL breast cancer cells gradually detached from the flask, growing progressively as spheroids. Phenotype was stable and reversible with cells remaining actively proliferating and easily accessible throughout culture. They could also be revived from frozen stocks. To achieve co-culture with fibroblasts in epiFL required use of low attachment culture vessels instead of standard plastic as fibroblasts remained adherent in epiFL. Here, cancer cell spheroids were allowed to form before adding fibroblasts. Immunohistochemical examination showed fibroblasts scattered throughout the epithelial spheroid, not dissimilar to the relationship of tumour stroma in human breast cancer. CONCLUSIONS: Because of its ease of handling, matrix-free 3D cell culture may be a useful model to study the influence of fibroblasts on breast cancer epithelial cells with use of epiFL culture medium taking this a step further towards a fully humanised 3D model. This methodology could be applied to other types of cancer cell lines, making this a versatile technique for cancer researchers wishing to use in vitro systems that better reflect cancer in vivo

CLIC3 controls recycling of late endosomal MT1-MMP and dictates invasion and metastasis in breast cancer

Chloride intracellular channel 3 (CLIC3) drives invasiveness of pancreatic and ovarian cancer by acting in concert with Rab25 to regulate the recycling of α5β1 integrin from late endosomes to the plasma membrane. Here, we show that in two estrogen receptor (ER)-negative breast cancer cell lines, CLIC3 has little influence on integrin recycling, but controls trafficking of the pro-invasive matrix metalloproteinase MT1-MMP (also known as MMP14). In MDA-MB-231 cells, MT1-MMP and CLIC3 are localized primarily to late endosomal/lysosomal compartments located above the plane of adhesion and near the nucleus. MT1-MMP is transferred from these late endosomes to sites of cell–matrix adhesion in a CLIC3-dependent fashion. Correspondingly, CLIC3-knockdown opposes MT1-MMP-dependent invasive processes. These include the disruption of the basement membrane as acini formed from MCF10DCIS.com cells acquire invasive characteristics in 3D culture, and the invasion of MDA-MB-231 cells into Matrigel or organotypic plugs of type I collagen. Consistent with this, expression of CLIC3 predicts poor prognosis in ER-negative breast cancer. The identification of MT1-MMP as a cargo of a CLIC3-regulated pathway that drives invasion highlights the importance of late endosomal sorting and trafficking in breast cancer

A multi-centre investigation towards reaching a consensus on the immunohistochemical detection of ER beta in archival formalin-fixed paraffin embedded human breast tissue

Estrogen receptor (ER) α is a well-established independent prognostic factor in breast cancer whose presence determines the clinical implications of adjuvant endocrine therapy. A second receptor, ERb has been described, and a number of studies have examined its expression in breast tissue. However elucidation of the role played by ERb has been hampered by published immunohistochemical studies employing a variety of protocols and scoring systems such that inter-laboratory comparisons are difficult. Here we present a multi-centre study designed to critically evaluate inter-laboratory differences in methodology. Six UK and Irish centres participated in this study. A small series of breast cancers were stained using centre-specific laboratory protocols and scored using both centrespecific and standard scoring protocols. There was generally poor agreement as to what constituted a positive or negative case when centre-specific scoring systems were used with less than half of all cases in agreement. Concordance was improved when a standard scoring system was used but varied according to threshold for positivity employed and primary antibody. Our results emphasise the need for further studies addressing the role of ERb to be based on a wider consensus on criteria for positivity. Ideally this should be based on calibration against clinical outcome

MCF-7 cells were plated on low adhesion plates and left for 48h undisturbed until spheroids formed.

<p>hTERT-immortalised fibroblasts transduced with dsRED, were then added and cocultured for a further 48h. Cells were imaged using a dual purpose phase contrast and fluorescence microscope, which allowed visualisation of cell distribution, defined by the fibroblasts’ red fluorescence and the MCF-7 cells’ lack of fluorescence under phase contrast. Scale bar = 400μm.</p

Immunohistochemical analysis of semi-serial sections of spheroid co-cultures.

<p>H&E staining of a representtaive spheroid is shown in (a). In (b) peripheral nuclear Ki67 immunoreactivity is seen, which seems to colocalise with epithelial cells, distinguished by cytokeratin 18 immunoreactivity, in an adjacent semi-serial section (c). Staining of a further semi-serial section with vimentin, used to detect fibroblasts shows fibroblast pockets scattered throughout the spheroid (d), not dissimilar to the relationship of tumour stroma in human breast cancer shown in the insert of (d), which illustrates a tissue microarray core stained with αSMA-stained stromal fibroblasts [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157004#pone.0157004.ref044" target="_blank">44</a>]. Positive immunoreactivity for each of the biomarkers is indicated by the brown staining. Scale bars = 100μm.</p