MicroRNA-196a promotes an oncogenic effect in head and neck cancer cells by suppressing annexin A1 and enhancing radioresistance.

Radiotherapy is a major treatment modality for head and neck squamous cell carcinoma (HNSCC). Up to 50% of patients with locally advanced disease relapse after radical treatment and there is therefore a need to develop predictive bomarkers for clinical use that allow the selection of patients who are likely to respond. MicroRNA (miRNA) expression profiling of a panel of HNSCC tumours with and without recurrent disease after surgery and radiotherapy detected miR-196a as one of the highest upregulated miRNAs in the poor prognostic group. To further study the role of miR-196a, its expression was determined in eight head and neck cancer cell lines. Overexpression of miR-196a in HNSCC cells, with low endogenous miR-196a expression, significantly increased cell proliferation, migration and invasion, and induced epithelial to mesenchymal transition. Conversely, miR-196a knockdown in cells with high endogenous expression levels significantly reduced oncogenic behaviour. Importantly, overexpression of miR-196a increased radioresistance of cells as measured by gamma H2AX staining and MTT survival assay. Annexin A1 (ANXA1), a known target of miR-196a, was found to be directly modulated by miR-196a as measured by luciferase assay and confirmed by Western blot analysis. ANXA1 knockdown in HNSCC exhibited similar phenotypic effects to miR-196a overexpression, suggesting the oncogenic effect of miR-196a may at least be partly regulated through suppression of ANXA1. In conclusion, this study identifies miR-196a as a potential important biomarker of prognosis and response of HNSCC to radiotherapy. Furthermore, our data suggest that miR-196a and/or its target gene ANXA1 could represent important therapeutic targets in HNSCC.

Apoptin interacts with and regulates the activity of protein kinase C beta in cancer cells.

Apoptin, the VP3 protein from chicken anaemia virus (CAV), induces tumour cell-specific cell death and represents a potential future anti-cancer therapeutic. In tumour but not in normal cells, Apoptin is phosphorylated and translocates to the nucleus, enabling its cytotoxic activity. Recently, the ß isozyme of protein kinase C (PKCß) was shown to phosphorylate Apoptin in multiple myeloma cell lines. However, the exact mechanism and nature of interaction between PKCß and Apoptin remain unclear. Here we investigated the physical and functional link between PKCß and CAV-Apoptin as well as with the recently identified Apoptin homologue derived from human Gyrovirus (HGyV). In contrast to HCT116 colorectal cancer cells the normal colon mucosa cell lines expressed low levels of PKCßI and showed reduced Apoptin activation, as evident by cytoplasmic localisation, decreased phosphorylation and lack of cytotoxic activity. Co-immunoprecipitation and proximity ligation assay studies identified binding of both CAV- and HGyV-Apoptin to PKCßI in HCT116 cells. Using Apoptin deletion constructs the N-terminal domain of Apoptin was found to be required for interacting with PKCßI. FRET-based PKC activity reporter assays by fluorescence lifetime imaging microscopy showed that expression of Apoptin in cancer cells but not in normal cells triggers a significant increase in PKC activity. Collectively, the results demonstrate a novel cancer specific interplay between Apoptin and PKCßI. Direct interaction between the two proteins leads to Apoptin-induced activation of PKC and consequently activated PKCßI mediates phosphorylation of Apoptin to promote its tumour-specific nuclear translocation and cytotoxic function.

Signalling of Apoptin.

The virus-derived protein Apoptin has the ability to induce p53-independent apoptosis in a variety of human cancer cells while leaving normal cells unharmed. It thus represents a potential anti-cancer therapeutic agent of the future but a proper understanding of Apoptin-induced signalling events is necessary prior to clinical application. The tumor-specific nuclear translocation and phosphorylation of Apoptin by a cellular kinase such as protein kinase C seem to be required for its function but otherwise the mode of tumor selectivity remains unknown. Apoptin has been shown to interact with several cellular proteins including Akt and the anaphase-promoting complex that regulate its activity and promote caspase-dependent apoptosis. This chapter summarizes the available data on tumor-specific pathways sensed by Apoptin and the mechanism of Apoptin-induced cell death.

Interleukin-7 and interleukin-15 drive CD4+CD28null T lymphocyte expansion and function in patients with acute coronary syndrome.

AIMS: Inflammation has important roles in atherosclerosis. CD4+CD28null (CD28null) T cells are a specialized T lymphocyte subset that produce inflammatory cytokines and cytotoxic molecules. CD28null T cells expand preferentially in patients with acute coronary syndrome (ACS) rather than stable angina and are barely detectable in healthy subjects. Importantly, ACS patients with CD28null T-cell expansion have increased risk for recurrent acute coronary events and poor prognosis, compared to ACS patients in whom this cell subset does not expand. The mechanisms regulating CD28null T-cell expansion in ACS remain elusive. We therefore investigated the role of cytokines in CD28null T-cell expansion in ACS. METHODS AND RESULTS: High-purity sorted CD4+ T cells from ACS patients were treated with a panel of cytokines (TNF-α, IL-1ß, IL-6, IL-7, and IL-15), and effects on the number, phenotype, and function of CD28null T cells were analysed and compared to the control counterpart CD28+ T-cell subset. IL-7- and IL-15-induced expansion of CD28null T cells from ACS patients, while inflammatory cytokines TNF-α, IL-1ß, and IL-6 did not. The mechanisms underlying CD28null T-cell expansion by IL-7/IL-15 were preferential activation and proliferation of CD28null T cells compared to control CD28+ T cells. Additionally, IL-7/IL-15 markedly augmented CD28null T-cell cytotoxic function and interferon-γ production. Further mechanistic analyses revealed differences in baseline expression of component chains of IL-7/IL-15 receptors (CD127 and CD122) and increased baseline STAT5 phosphorylation in CD28null T cells from ACS patients compared to the control CD28+ T-cell subset. Notably, we demonstrate that CD28null T-cell expansion was significantly inhibited by Tofacitinib, a selective JAK1/JAK3 inhibitor that blocks IL-7/IL-15 signalling. CONCLUSION: Our novel data show that IL-7 and IL-15 drive the expansion and function of CD28null T cells from ACS patients suggesting that IL-7/IL-15 blockade may prevent expansion of these cells and improve patient outcomes.

The DebugIT core ontology: semantic integration of antibiotics resistance patterns.

Antibiotics resistance development poses a significant problem in today's hospital care. Massive amounts of clinical data are being collected and stored in proprietary and unconnected systems in heterogeneous format. The DebugIT EU project promises to make this data geographically and semantically interoperable for case-based knowledge analysis approaches aiming at the discovery of patterns that help to align antibiotics treatment schemes. The semantic glue for this endeavor is DCO, an application ontology that enables data miners to query distributed clinical information systems in a semantically rich and content driven manner. DCO will hence serve as the core component of the interoperability platform for the DebugIT project. Here we present DCO and an approach thet uses the semantic web query language SPARQL to bind and ontologically query hospital database content using DCO and information model mediators. We provide a query example that indicates that ontological querying over heterogeneous information models is feasible via SPARQL construct- and resource mapping queries.

TLR9 Mediated Tumor-Stroma Interactions in Human Papilloma Virus (HPV)-Positive Head and Neck Squamous Cell Carcinoma Up-Regulate PD-L1 and PD-L2.

Background: The co-inhibitory receptor PD-1 is expressed in many tumors including head and neck squamous cell carcinoma (HNSCC) and is an important immunotherapy target. However, the role of PD-1 ligands, PD-L1, and particularly PD-L2, in the tumor-stromal cell interactions that cause a tumor-permissive environment in HNSCC is not completely understood and is the focus of our study. Methods: Expression of PD-L1 and PD-L2 was analyzed by immunohistochemistry in situ in HNSCC tumor tissue. Co-cultures were established between stromal cells (fibroblasts and macrophages) and human papilloma virus (HPV)-positive and HPV-negative HNSCC cell lines (HNSCCs) and PD-1 ligands expression was analyzed using flow cytometry. Results: PD-L1 and PD-L2 were expressed both in tumor cells and stroma in HNSCC tissue in situ. In vitro, basal expression of PD-L1 and PD-L2 was low in HNSCCs and high on fibroblasts and macrophages. Interestingly, HPV-positive but not HPV-negative HNSCCs increased the expression of both PD-1 ligands on fibroblasts upon co-culture. This effect was not observed with macrophages. Conversely, both fibroblasts and macrophages increased PD-1 ligands on HPV-positive HNSCCs, whilst this was not observed in HPV-negative HNSCCs. Crucially, we demonstrate that up-regulation of PD-L1 and PD-L2 on fibroblasts by HPV-positive HNSCCs is mediated via TLR9. Conclusions: This work demonstrates in an in vitro model that HPV-positive HNSCCs regulate PD-L1/2 expression on fibroblasts via TLR9. This may open novel avenues to modulate immune checkpoint regulator PD-1 and its ligands by targeting TLR9.

Registered report: Systematic identification of genomic markers of drug sensitivity in cancer cells.

The Reproducibility Project: Cancer Biology seeks to address growing concerns about the reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Systematic identification of genomic markers of drug sensitivity in cancer cells" by Garnett and colleagues, published in Nature in 2012 (Garnett et al., 2012). The experiments to be replicated are those reported in Figures 4C, 4E, 4F, and Supplemental Figures 16 and 20. Garnett and colleagues performed a high throughput screen assessing the effect of 130 drugs on 639 cancer-derived cell lines in order to identify novel interactions for possible therapeutic approaches. They then tested this approach by exploring in more detail a novel interaction they identified in which Ewing's sarcoma cell lines showed an increased sensitivity to PARP inhibitors (Figure 4C). Mesenchymal progenitor cells (MPCs) transformed with the signature EWS-FLI1 translocation, the hallmark of Ewing's sarcoma family tumors, exhibited increased sensitivity to the PARP inhibitor olaparib as compared to MPCs transformed with a different translocation (Figure 4E). Knockdown mediated by siRNA of EWS-FLI1 abrogated this sensitivity to olaparib (Figure 4F). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

Targeting T cells to treat atherosclerosis: odyssey from bench to bedside.

More than 150 years from the initial description of inflammation in atherosclerotic plaques, randomized clinical trials to test anti-inflammatory therapies in atherosclerosis have recently been initiated. Lymphocytes and macrophages are main participants in the inflammatory response in atherosclerosis. T lymphocytes operate mainly by exerting strong influences on the function of many cells in the immune system and beyond, and co-ordinating their interactions. Importantly, T lymphocytes are not a homogenous population, but include several subsets with specialized functions that can either promote or suppress inflammation. The interactions between these T-lymphocyte subsets have critical consequences on the course and outcome of inflammation. The complexity of the inflammatory response in atherosclerosis poses significant challenges on translating experimental findings into clinical therapies and makes the journey from bench to bedside an arduous one. Here, we summarize recent advances on the role of CD4(+) T cells in the inflammatory process in atherosclerosis and discuss potential therapies to modulate these lymphocytes that may provide future breakthroughs in the treatment of atherosclerosis.