Modulation of the inwardly rectifying potassium channel Kir4.1 by the pro-invasive miR-5096 in glioblastoma cells.

IF 5.168International audienceInwardly rectifying potassium channels (Kir), and especially the barium-sensitive Kir4.1 encoded by KCNJ10, are key regulators of glial functions. A lower expression or mislocation of Kir4.1 is detected in human brain tumors. MicroRNAs participate in the regulation of ionic channels and associated neurologic disorders. Here, we analyze effects of miR-5096 on the Kir4.1 expression and function in two glioblastoma cell lines, U87 and U251. Using whole-cell patch-clamp and western-blot analysis, we show that cell loading with miR-5096 decreases the Kir4.1 protein level and associated K+ current. Cell treatment with barium, a Kir4.1 blocker, or cell loading of miR-5096 both increase the outgrowth of filopodia in glioma cells, as observed by time-lapse microscopy. Knocking-down Kir4.1 expression by siRNA transfection similarly increased both filopodia formation and invasiveness of glioma cells as observed in Boyden chamber assay. MiR-5096 also promotes the release of extracellular vesicles by which it increases its own transfer to surrounding cells, in a Kir4.1-dependent manner in U251 but not in U87. Altogether, our results validate Kir4.1 as a miR-5096 target to promote invasion of glioblastoma cells. Our data highlight the complexity of microRNA effects and the role of K+ channels in cancer

Data from: Improving access and continuity of care for homeless people: how could general practitioners effectively contribute? results from a mixed study.

Objectives: To analyze the views of general practitioners (GPs) about how they can provide care to homeless people (HP) and to explore which measures could influence their views. Design: Mixed-methods design (qualitative –> quantitative (cross-sectional observational) qualitative). Qualitative data were collected through semi-structured interviews; quantitative data were collected through questionnaires with closed questions. Quantitative data were analyzed with descriptive statistical analyses on SPPS; a content analysis was applied on qualitative data. Setting: primary care; views of urban GPs working in deprived area in Marseille were explored by questionnaires and/or semi-structured interview. Participants: 19 GPs involved in HP’s healthcare were recruited for phase 1 (qualitative); for phase 2 (quantitative), 150 GPs who provide routine health care (“standard” GPs) were randomized, 144 met the inclusion criteria and 105 responded the questionnaire; for phase 3 (qualitative), data were explored on 14 “standard” GPs. Results: In quantitative phase, 79% of the 105 GPs already treated HP. Most of the difficulties they encountered treating HP concerned social matters (mean level of perceived difficulties = 3.95/5, IC95 [3.74-4.17]), lack of medical information (mn=3.78/5, IC95 [3.55-4.01]patient’s compliance (mn=3.67/5, IC95 [3.45-3.89]), loneliness in practice (mn=3.45/5, IC95 [3.18-3.72]) and time required for doctor (mn=3.25, IC95 [3-3.5]. From qualitative analysis we understood that maintaining a stable follow-up was a major condition for GPs to contribute effectively to the care of HP. Acting on health system organization, developing a medical and psychosocial approach with closer relation with social workers and enhancing the collaboration between tailored and non-tailored programs were also other key answers. Conclusion: If we adapt the conditions of GPs practice, they could contribute to the improvement of HP’s health. These results will enable the construction of a new model of primary care organization aiming to improve access to health care for HP

Annex 2 - extracts from coding tree in english

Extracts from the coding trees (non-exhaustive) used for qualitative phases (phases 1 and 3), from N-Vivo 11

Improving access and continuity of care for homeless people: how could ă general practitioners effectively contribute? Results from a mixed study

International audienceObjectives: To analyse the views of general practitioners (GPs) about ă how they can provide care to homeless people (HP) and to explore which ă measures could influence their views. ă Design: Mixed-methods design (qualitative -> quantitative ă (cross-sectional observational) -> qualitative). Qualitative data were ă collected through semistructured interviews and through questionnaires ă with closed questions. Quantitative data were analysed with descriptive ă statistical analyses on SPPS; a content analysis was applied on ă qualitative data. ă Setting: Primary care; views of urban GPs working in a deprived area in ă Marseille were explored by questionnaires and/or semistructured ă interview. ă Participants: 19 GPs involved in HP's healthcare were recruited for ă phase 1 (qualitative); for phase 2 (quantitative), 150 GPs who provide ă routine healthcare ('standard' GPs) were randomised, 144 met the ă inclusion criteria and 105 responded to the questionnaire; for phase 3 ă (qualitative), data were explored on 14 `standard' GPs. ă Results: In the quantitative phase, 79% of the 105 GPs already treated ă HP. Most of the difficulties they encountered while treating HP ă concerned social matters (mean level of perceived difficulties=3.95/5, ă IC 95 (3.74 to 4.17)), lack of medical information (mn=3.78/5, IC 95 ă (3.55 to 4.01)) patient's compliance (mn=3.67/5, IC 95 (3.45 to 3.89)), ă loneliness in practice (mn=3.45/5, IC 95 (3.18 to 3.72)) and time ă required for the doctor (mn=3.25, IC 95 (3 to 3.5)). From qualitative ă analysis we understood that maintaining a stable follow-up was a major ă condition for GPs to contribute effectively to the care of HP. Acting on ă health system organisation, developing a medical and psychosocial ă approach with closer relation with social workers and enhancing the ă collaboration between tailored and non-tailored programmes were also ă other key answers. ă Conclusions: If we adapt the conditions of GPs practice, they could ă contribute to the improvement of HP's health. These results will enable ă the construction of a new model of primary care organisation aiming to ă improve access to healthcare for HP

Transfer of functional microRNAs between glioblastoma and microvascular endothelial cells through gap junctions

IF 5.008International audienceExtensive invasion and angiogenesis are hallmark features of malignant glioblastomas. Here, we co-cultured U87 human glioblastoma cells and human microvascular endothelial cells (HMEC) to demonstrate the exchange of microRNAs that initially involve the formation of gap junction communications between the two cell types. The functional inhibition of gap junctions by carbenoxolone blocks the transfer of the anti-tumor miR-145-5p from HMEC to U87, and the transfer of the pro-invasive miR-5096 from U87 to HMEC. These two microRNAs exert opposite effects on angiogenesis in vitro. MiR-5096 was observed to promote HMEC tubulogenesis, initially by increasing Cx43 expression and the formation of heterocellular gap junctions, and secondarily through a gap-junction independent pathway. Our results highlight the importance of microRNA exchanges between tumor and endothelial cells that in part involves the formation of functional gap junctions between the two cell types

Abstract LB-017: HSP110 sustains aberrant NFkB signaling in activated B-cell diffuse large B-cell lymphoma through MyD88 stabilization

International audienceDiffuse large B cell lymphoma (DLBCL) is an aggressive lymphoproliferative disorder of B lymphocytes accounting for 30 % of adult Non Hodgkin Lymphoma (NHL). Among DLBCL, Activated B Cell - DLBCL (ABC-DLBCL) is the most aggressive form and has a poor prognosis. Heat-shock proteins (HSPs) are molecular chaperons highly expressed in cancer cells and implicated in resistance to radio- and chemotherapy. Therefore, HSPs are envisioned as therapeutic targets in many cancers. Among the different HSPs, HSP110 has been recently identified as a pro-survival factor in germinal center-derived DLBCL (GC-DLBCL), through stabilization of the GC-DLBCL oncogene Bcl-6. Here, we have explored if HSP110 could also be involved in the survival of the most aggressive form of DLBCL. We observed a high HSP110 expression in all ABC-DLBCL patient samples, compared to normal reactive lymph nodes by using IHC staining of ABC-DLBCL tumor sections and transcriptional analysis of ABC-DLBCL patient tumors. Furthermore, shRNA silencing of HSP110 decreases the survival of several ABC-DLBCL cell lines, and downregulates the expression of pro-survival factors such as BcL2 and BcL-XL. SiRNA silencing of HSP110 abrogates NF-kB signaling, which is the major oncogenic pathway in ABC-DLBCL cell lines. In accord with these results, over-expression of HSP110 in DLBCL and non-DLBCL cell lines increases NF-kB signaling, indicating a tight interplay between HSP110 and the NF-kB pathway. Using immune-precipitation and DuolinkTM assays, we identified an in vitro and in cellulo interaction between HSP110 and Myd88, a critical protein of the NF-kB pathway that bears an activated mutation in many ABC-DLBC patients and that is responsible for lymphoma aggressiveness. Finally, we demonstrate that HSP110 stabilizes the wild type as well as the mutated form of Myd88, therefore facilitating the chronic NF-kB pathway activation in those cells. In conclusion, we identified HSP110 as a regulator of NF-kB signaling through MyD88 stabilization in ABC-DLBCL. This finding highlights HSP110 as a new potential therapeutic target in DLBCL

The biofilm mode of life boosts the anti-inflammatory properties of Lactobacillus

The predominant form of life for microorganisms in their natural habitats is the biofilm mode of growth. The adherence and colonization of probiotic bacteria are considered as essential factors for their immunoregulatory function in the host. Here, we show that Lactobacillus caseiATCC334 adheres to and colonizes the gut of zebrafish larvae. The abundance of pro-inflammatory cytokines and the recruitment of macrophages were low when inflammation was induced in probiotic-fed animals, suggesting that these bacteria have anti-inflammatory properties. We treated human macrophage-differentiated monocytic THP-1 cells with supernatants of L.caseiATCC334 grown in either biofilm or planktonic cultures. TNF- production was suppressed and the NF-B pathway was inhibited only in the presence of supernatants from biofilms. We identified GroEL as the biofilm supernatant compound responsible, at least partially, for this anti-inflammatory effect. Gradual immunodepletion of GroEL demonstrated that the abundance of GroEL and TNF- were inversely correlated. We confirmed that biofilm development in other Lactobacillus species affects the immune response. The biofilms supernatants of these species also contained large amounts of GroEL. Thus, our results demonstrate that the biofilm enhances the immunomodulatory effects of Lactobacillus sp. and that secreted GroEL is involved in this beneficial effect

Extracellular HSP110 skews macrophage polarization in colorectal cancer

IF 7.644International audienceHSP110 is induced by different stresses and, through its anti-apoptotic and chaperoning properties, helps the cells to survive these adverse situations. In colon cancers, HSP110 is abnormally abundant. We have recently showed that colorectal cancer (CRC) patients with microsatellite instability (MSI) had an improved response to chemotherapy because they harbor an HSP110 inactivating mutation (HSP110DE9). In this work, we have used patients' biopsies and human CRC cells grown in vitro and in vivo (xenografts) to demonstrate that (1) HSP110 is secreted by CRC cells and that the amount of this extracellular HSP110 is strongly decreased by the expression of the mutant HSP110DE9, (2) Supernatants from CRC cells overexpressing HSP110 or purified recombinant human HSP110 (LPS-free) affect macrophage differentiation/polarization by favoring a pro-tumor, anti-inflammatory profile, (3) Conversely, inhibition of HSP110 (expression of siRNA, HSP110DE9 or immunodepletion) induced the formation of macrophages with a cytotoxic, pro-inflammatory profile. (4) Finally, this effect of extracellular HSP110 on macrophages seems to implicate TLR4. These results together with the fact that colorectal tumor biopsies with HSP110 high were infiltrated with macrophages with a pro-tumoral profile while those with HSP110 low were infiltrated with macrophages with a cytotoxic profile, suggest that the effect of extracellular HSP110 function on macrophages may also contribute to the poor outcomes associated with HSP110 expression

Regulation of the proapoptotic functions of prostate apoptosis response-4 (Par-4) by casein kinase 2 in prostate cancer cells

International audienceThe proapoptotic protein, prostate apoptosis response-4 (Par-4), acts as a tumor suppressor in prostate cancer cells. The serine/threonine kinase casein kinase 2 (CK2) has a well-reported role in prostate cancer resistance to apoptotic agents or anticancer drugs. However, the mechanistic understanding on how CK2 supports survival is far from complete. In this work, we demonstrate both in rat and humans that (i) Par-4 is a new substrate of the survival kinase CK2 and (ii) phosphorylation by CK2 impairs Par-4 proapoptotic functions. We also unravel different levels of CK2-dependent regulation of Par-4 between species. In rats, the phosphorylation by CK2 at the major site, S124, prevents caspase-mediated Par-4 cleavage (D123) and consequently impairs the proapoptotic function of Par-4. In humans, CK2 strongly impairs the apoptotic properties of Par-4, independently of the caspase-mediated cleavage of Par-4 (D131), by triggering the phosphorylation at residue S231. Furthermore, we show that human Par-4 residue S231 is highly phosphorylated in prostate cancer cells as compared with their normal counterparts. Finally, the sensitivity of prostate cancer cells to apoptosis by CK2 knockdown is significantly reversed by parallel knockdown of Par-4. Thus, Par-4 seems a critical target of CK2 that could be exploited for the development of new anticancer drugs