María Salazar

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that delta(9)-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.

The transcription factor Nrf2 (nuclear factor E2-related factor 2) regulates the expression of antioxidant phase II genes and contributes to preserve redox homeostasis and cell viability in response to oxidant insults. Nrf2 should be coordinated with the canonical cell survival pathway represented by phosphatidylinositol 3-kinase (PI3K) and the Ser/Thr kinase Akt but so far the mechanistic connections remain undefined. Here we identify glycogen synthase kinase-3β (GSK-3β), which is inhibited by Akt-mediated phosphorylation, as the link between both processes. Using heme oxygenase-1 (HO-1) as a model phase II gene, we found that both PI3K and Akt increased mRNA and protein levels of this enzyme. Pharmacological inhibitors (LiCl and PDZD-8) and genetic variants of GSK-3β (constitutively active and dominant negative mutants) indicated that PI3K/Akt activates and GSK-3β inhibits the antioxidant response elements of the <i>ho1</i> promoter and pointed Nrf2 as directly involved in this process. Indeed, GSK-3β phosphorylated Nrf2 <i>in vitro</i> and <i>in vivo</i>. Immunocytochemistry and subcellular fractionation analyses demonstrated that the effect of GSK-3β-mediated phosphorylation of Nrf2 is to exclude this transcription factor from the nucleus. Nrf2 up-regulated the expression of HO-1, glutathione peroxidase, glutathione <i>S</i>-transferase A1, NAD(P)H: quinone oxidoreductase and glutamate-cysteine ligase and protected against hydrogen peroxide-induced glutathione depletion and cell death, whereas co-expression of active GSK-3β attenuated both phase II gene expression and oxidant protection. These results contribute to clarify the cross-talk between the survival signal elicited by PI3K/Akt and the antioxidant phase II cell response, and introduce GSK-3β as the key mediator of this regulation mechanism.

Different physiological and pathological conditions can perturb protein folding in the endoplasmic reticulum, leading to a condition known as ER stress. ER stress activates a complex intracellular signal transduction pathway, called unfolded protein response (UPR). The UPR is tailored essentially to reestablish ER homeostasis also through adaptive mechanisms involving the stimulation of autophagy. However, when persistent, ER stress can switch the cytoprotective functions of UPR and autophagy into cell death promoting mechanisms. Recently, a variety of anticancer therapies have been linked to the induction of ER stress in cancer cells, suggesting that strategies devised to stimulate its prodeath function or block its prosurvival function, could be envisaged to improve their tumoricidial action. A better understanding of the molecular mechanisms that determine the final outcome of UPR and autophagy activation by chemotherapeutic agents, will offer new opportunities to improve existing cancer therapies as well as unravel novel targets for cancer treatment.

Glioblastoma multiforme (GBM) is highly resistant to current anticancer treatments, which makes it crucial to find new therapeutic strategies aimed at improving the poor prognosis of patients suffering from this disease. Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoid receptor agonists inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, we show that the combined administration of THC and temozolomide (TMZ; the benchmark agent for the management of GBM) exerts a strong antitumoral action in glioma xenografts, an effect that is also observed in tumors that are resistant to TMZ treatment. Combined administration of THC and TMZ enhanced autophagy, whereas pharmacologic or genetic inhibition of this process prevented TMZ + THC-induced cell death, supporting that activation of autophagy plays a crucial role on the mechanism of action of this drug combination. Administration of submaximal doses of THC and cannabidiol (CBD; another plant-derived cannabinoid that also induces glioma cell death through a mechanism of action different from that of THC) remarkably reduces the growth of glioma xenografts. Moreover, treatment with TMZ and submaximal doses of THC and CBD produced a strong antitumoral action in both TMZ-sensitive and TMZ-resistant tumors. Altogether, our findings support that the combined administration of TMZ and cannabinoids could be therapeutically exploited for the management of GBM.

Article2 May 2017free access Source DataTransparent process Programmed mitophagy is essential for the glycolytic switch during cell differentiation Lorena Esteban-Martínez Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Elena Sierra-Filardi Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Rebecca S McGreal Departments of Genetics, Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author María Salazar-Roa Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Guillermo Mariño Departamento de Biología Fundamental, Universidad de Oviedo, Fundación para la Investigación Sanitaria del Principado de Asturias (FINBA), Oviedo, Spain Search for more papers by this author Esther Seco Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Sylvère Durand Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France Search for more papers by this author David Enot Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France Search for more papers by this author Osvaldo Graña Bioinformatics Unit and Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marcos Malumbres orcid.org/0000-0002-0829-6315 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Ales Cvekl Departments of Genetics, Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Ana María Cuervo orcid.org/0000-0002-0771-700X Department of Developmental and Molecular Biology, Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Guido Kroemer Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France Equipe 11 labellisée par la Ligue Nationale contre le cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Université Pierre et Marie Curie, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Department of Women's and Children's Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Patricia Boya Corresponding Author [email protected] orcid.org/0000-0003-3045-951X Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Lorena Esteban-Martínez Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Elena Sierra-Filardi Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Rebecca S McGreal Departments of Genetics, Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author María Salazar-Roa Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Guillermo Mariño Departamento de Biología Fundamental, Universidad de Oviedo, Fundación para la Investigación Sanitaria del Principado de Asturias (FINBA), Oviedo, Spain Search for more papers by this author Esther Seco Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Sylvère Durand Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France Search for more papers by this author David Enot Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France Search for more papers by this author Osvaldo Graña Bioinformatics Unit and Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marcos Malumbres orcid.org/0000-0002-0829-6315 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Ales Cvekl Departments of Genetics, Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Ana María Cuervo orcid.org/0000-0002-0771-700X Department of Developmental and Molecular Biology, Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, NY, USA Search for more papers by this author Guido Kroemer Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France Equipe 11 labellisée par la Ligue Nationale contre le cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Université Pierre et Marie Curie, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Department of Women's and Children's Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden Search for more papers by this author Patricia Boya Corresponding Author [email protected] orcid.org/0000-0003-3045-951X Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain Search for more papers by this author Author Information Lorena Esteban-Martínez1, Elena Sierra-Filardi1, Rebecca S McGreal2, María Salazar-Roa3, Guillermo Mariño4, Esther Seco1, Sylvère Durand5, David Enot5, Osvaldo Graña6, Marcos Malumbres3, Ales Cvekl2, Ana María Cuervo7, Guido Kroemer5,8,9,10,11,12,13 and Patricia Boya *,1 1Department of Cellular and Molecular Biology, Centro de Investigaciones Biológicas, CSIC, Madrid, Spain 2Departments of Genetics, Ophthalmology and Visual Sciences, Albert Einstein College of Medicine, Bronx, NY, USA 3Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 4Departamento de Biología Fundamental, Universidad de Oviedo, Fundación para la Investigación Sanitaria del Principado de Asturias (FINBA), Oviedo, Spain 5Metabolomics and Molecular Cell Biology Platforms, Gustave Roussy, Villejuif, France 6Bioinformatics Unit and Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 7Department of Developmental and Molecular Biology, Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, NY, USA 8Equipe 11 labellisée par la Ligue Nationale contre le cancer, Centre de Recherche des Cordeliers, Paris, France 9INSERM, U1138, Paris, France 10Université Paris Descartes, Sorbonne Paris Cité, Paris, France 11Université Pierre et Marie Curie, Paris, France 12Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 13Department of Women's and Children's Health, Karolinska Institute, Karolinska University Hospital, Stockholm, Sweden *Corresponding author. Tel: +34 91 8373112 Ext 4369; E-mail: [email protected] EMBO J (2017)36:1688-1706https://doi.org/10.15252/embj.201695916 See also: A Deczkowska & M Schwartz (June 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Retinal ganglion cells (RGCs) are the sole projecting neurons of the retina and their axons form the optic nerve. Here, we show that embryogenesis-associated mouse RGC differentiation depends on mitophagy, the programmed autophagic clearance of mitochondria. The elimination of mitochondria during RGC differentiation was coupled to a metabolic shift with increased lactate production and elevated expression of glycolytic enzymes at the mRNA level. Pharmacological and genetic inhibition of either mitophagy or glycolysis consistently inhibited RGC differentiation. Local hypoxia triggered expression of the mitophagy regulator BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L, best known as NIX) at peak RGC differentiation. Retinas from NIX-deficient mice displayed increased mitochondrial mass, reduced expression of glycolytic enzymes and decreased neuronal differentiation. Similarly, we provide evidence that NIX-dependent mitophagy contributes to mitochondrial elimination during macrophage polarization towards the proinflammatory and more glycolytic M1 phenotype, but not to M2 macrophage differentiation, which primarily relies on oxidative phosphorylation. In summary, developmentally controlled mitophagy promotes a metabolic switch towards glycolysis, which in turn contributes to cellular differentiation in several distinct developmental contexts. Synopsis Retinal ganglion cell differentiation and M1 macrophage polarization depend on metabolic reprogramming towards glycolysis, which is triggered by hypoxia-induced autophagic degradation of mitochondria (mitophagy). Programmed mitophagy eliminates mitochondria during mouse retinal development. Hypoxia-induces NIX-dependent mitophagy. Mitophagy allows for a glycolytic shift required for retinal ganglion cell differentiation. Mitophagy also regulates metabolic reprogramming during M1 macrophage polarization. Introduction Autophagy is a catabolic pathway that mediates the degradation and recycling of intracellular components, including whole organelles, to sustain cell homeostasis (Boya et al, 2013). Autophagy is the sole mechanism that allows for the degradation of entire mitochondria, a process commonly known as mitophagy. While mitophagy primarily eliminates damaged or dysfunctional mitochondria (Ashrafi & Schwarz, 2013), this pathway can also mediate the degradation of mitochondria in developmental contexts, a process known as programmed mitophagy (Ney, 2015). BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L), also known as NIX, is an essential regulator of programmed mitophagy during reticulocyte maturation (Aerbajinai et al, 2003; Diwan et al, 2007; Schweers et al, 2007; Sandoval et al, 2008). During programmed mitophagy, NIX is mainly regulated at the transcriptional level and acts as a mitophagy receptor, mediating autophagic sequestration of mitochondria by interacting with LC3 via its LIR domain (Novak et al, 2010). Metabolic reprogramming is a process by which cells shift their metabolism from oxidative phosphorylation towards glycolysis and convert glucose into lactate even in the presence of oxygen. This phenomenon was first described in cancer cells, but has since been observed in other cell types, including embryonic stem cells, proinflammatory M1 macrophages and cells of the adult retina, and it is thought to be essential to fulfill the metabolic requirements of those cells (Galvan-Pena & O'Neill, 2014; Ng et al, 2015; Chandel et al, 2016). Hypoxia also triggers metabolic shift towards glycolysis. This type of metabolism is also observed in stem cells and is thought to constitute an adaptation to the hypoxic conditions present in vivo during development, in adult stem cell niches and in the inflamed tissue (Suda et al, 2011; Escribese et al, 2012). Interestingly, hypoxia is a strong inductor of mitophagy (Zhang et al, 2008). How the complex functionality of the nervous system arises from a pool of undifferentiated neuroepithelial cells is one of the more fascinating aspects of embryonic development (Valenciano et al, 2008). These neuroepithelial cells undergo marked changes to generate differentiated neurons and glial cells. Cell proliferation requires nutrients, energy and biosynthetic activity to duplicate all macromolecular components during each passage of the cell cycle. Differentiation requires profound changes in cellular components and a shift in metabolic activity. Indeed, metabolic pathways may be controlled by the same signals that control cell differentiation (Agathocleous & Harris, 2013). The retina, a model organ for the study of the central nervous system, is a three-layered structure composed of one glial and six distinct neuronal cell types, which arise from a pool of multipotent retinal progenitor cells (Stenkamp, 2015). In mice, retinal neurogenesis is an orderly process that starts with the differentiation of retinal ganglion cells (RGCs) and is followed by that of other neuronal cell types. During retinal development, cell differentiation follows a central-to-peripheral gradient, with the result that the central retina is more developmentally advanced than the peripheral retina (Stenkamp, 2015). We have previously shown that autophagy genes are essential for the generation of mature neurons from olfactory bulb neural stem cells (Vazquez et al, 2012). Moreover, blockade of autophagy in the embryonic chick retina results in decreased ATP levels and hampers the elimination of apoptotic cells generated during neurogenesis (Mellén et al, 2008, 2009). Interestingly, both these phenotypes are restored by supplying pyruvate as a cell-permeable precursor (methyl pyruvate), suggesting a connection between autophagy and cell metabolism during neuronal differentiation (Boya et al, 2016). Moreover, cancer cells engage mitophagy to promote metabolic reprogramming towards glycolysis during prolonged mitotic arrest (Domenech et al, 2015). Here, we show that NIX- and Atg5-dependent mitophagy mediates a metabolic shift towards glycolysis that allows RGC generation in the embryonic mouse retina. Furthermore, we describe a mitophagy-dependent metabolic shift that occurs during the polarization of macrophages towards a proinflammatory and more glycolytic M1 phenotype. Taken together, our data show that programmed mitophagy triggers metabolic reprogramming towards glycolysis during cellular differentiation. Results Mitochondrial rarefaction during embryonic RGC differentiation In the mouse retina, neurogenesis starts around day E12.5 with the differentiation of RGCs, the first neurons to be generated. As expected, immunofluorescence analyses revealed the presence of the RGC-specific transcription factor Brn3a and γ-synuclein (another RGC marker) from E13.5, with levels peaking at E15.5 in the RGC layer of mouse retinal flatmounts (Fig 1A and B, and Appendix Figs S1 and S2). In the same retinas, immunostaining for the mitochondrial protein TOMM20 decreased after E13.5 (Fig 1C and D, insets below, and Appendix Fig S1C). This decrease in the number of mitochondria during retinal embryonic development was corroborated by labelling dissociated retinas at different embryonic stages with the mitochondrion-specific dye MitoTracker Deep Red (MTDR), which was quantified by flow cytometry (Fig 1E and F). This decrease in mitochondrial number could not be attributed to decreased expression of genes involved in mitochondrial biogenesis, such as Ppargc1a and Tfam, as their expression remained unchanged during the embryonic stages assessed (Fig EV1A). Moreover, electron microscopy analyses revealed that at E15.5 the number of mitochondria in each RGC was reduced as compared with that of proliferating neuroblasts (Fig 1G and H). Mitochondria from neuroblasts (Nbs) were large and displayed well-organized cristae, indicative of a high metabolic rate, while those from RGCs were comparatively smaller and contained fewer cristae (Fig 1I). Together these data suggest that programmed elimination of mitochondria occurs during RGC development. Figure 1. Mitochondrial mass decreases during embryonic retinal development A. Immunostaining of the retinal ganglion cell (RCG)-specific transcription factor Brn3a (cyan) in the RGC layer of mouse retinal flatmounts at the indicated embryonic days (E). The maximal projection in the RGC layer is shown. Scale bar, 50 μm. B. Quantification of RGC density per mm2 at the indicated embryonic stages in retinas stained as in (A) (n = 6 per group). Data are presented as mean ± SEM. ***P < 0.001 (Mann-Whitney U-test). C. Immunostaining with the mitochondrial marker TOMM20 (red) in the RGC layer of the same retinas as in (A). Scale bar, 50 μm. Insets show TOMM20 staining (red) and nuclei labelled with DAPI (blue) in a confocal plane in the indicated region. Scale bar, 20 μm. D. TOMM20-positive pixel staining at the indicated embryonic stages labelled as in (C) (n = 6 per group). Data are presented as mean ± SEM. ***P < 0.001 (Mann-Whitney U-test). E, F. Flow cytometry histograms and percentage mean fluorescence intensity (% MitoTracker) in dissociated retinas, corresponding to different embryonic stages, stained with MitoTracker Deep Red (MTDR) (n = 5–12 retinas per group). Data are presented as mean ± SEM. **P < 0.01, ***P < 0.001 (Mann-Whitney U-test). G. Ultrastructural analysis of neuroblast (Nb) and RGC from an E15.5 mouse retina. Scale bar, 1 μm. Arrows indicate mitochondria. H. Quantification of the number of mitochondria per cell in G (n = 12–14 cells per group). Data are presented as mean ± SEM. *P < 0.05 (Mann-Whitney U-test). I. Mitochondrial morphology (m) in retinal Nb and RGC at E15.5. Scale bars, 500 nm. J. Immunoblot of retinas at different embryonic stages incubated for 3 h in the absence (−) or presence (+) of hydroxychloroquine to block autophagic flux. K. Autophagosomal membrane (AP) surrounding a mitochondrion (arrows in left-hand image) in an E15.5 mouse retina in the presence of HCQ to block lysosomal degradation. Scale bar, 500 nm. Insets of the indicated areas show four adjacent membranes (right). L. Increase in the number of mitochondrial per nm2 in RGCs from E15.5 mouse retinas in the presence of HCQ (n = 12 per group). Data are presented as mean ± SEM. ***P < 0.001 (Mann-Whitney U-test). Source data are available online for this figure. Source Data for Figure 1 [embj201695916-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The increase in mitochondrial number is autophagy- and mitophagy dependent mRNA expression, determined by qRT–PCR, of the mitochondrial biogenesis regulators Ppargc1a and Tfam (n = 2–3 pools of retinas per group). Data are presented as mean ± SEM. TOMM20 immunostaining at the indicated developmental stages in flat-mounted retinas incubated for 6 h in the presence of 5 μM CsA or 10 mM 3-MA. The maximal projection of the z-stack is shown. Scale bar, 50 μm. COX-IV immunostaining in flat-mounted retinas (E15.5) incubated for 6 h with 3-MA or CsA. Maximal projections are shown on the left (scale bar, 50 μm) and single confocal planes from the boxed insets are shown on the right. COX-IV immunostaining is shown in green and DAPI-stained nuclei in blue. Scale bar in insets, 20 μm. COX-IV immunoblotting of E15.5 retinas incubated for 6 h in 3-MA or CsA. Tubulin was used as a loading control. Percentage of mean fluorescence intensity (% MitoTracker) in E15.5 retinas incubated with 3-MA, CsA or a combination of both (n = 6–22 retinas per group). Data are presented as mean ± SEM. *P < 0.05 (Student's t-test). Source data are available online for this figure. Download figure Download PowerPoint Mitophagy-mediated elimination of mitochondria during RGC development Since autophagy is the only known catabolic pathway capable of mediating the degradation of entire mitochondria, we analysed autophagic activity during embryonic development in the mouse retina. The autophagy-associated lipidation of microtubule-associated proteins 1A/1B light chain 3 (MAP1LC3, better known as LC3), which gives rise to an increase in the electrophoretic mobility of LC3 (LC3-II), was increased during retinal development (Fig 1J). Consistently, incubation of retinal explants for 3 h with the lysosomal inhibitor hydroxychloroquine (HCQ) further increased LC3-II accumulation, indicating that basal autophagic flux is enhanced during the early stages of development of the embryonic mouse retina (Fig 1J). Transmission electron microscopy revealed the presence of mitochondria inside autophagosomes in RGCs at E15.5 in these conditions (Fig 1K) and the number of mitochondria was increased after inhibition of lysosomal degradation (Fig 1L), suggesting that the decrease in mitochondrial mass during retinal development is mediated by autophagy. Next, we incubated retinas at different embryonic stages with the general autophagy inhibitor 3-methyladenine (3-MA) or with cyclosporine A (CsA), an inhibitor of mitochondrial cyclophilin D that suppresses mitophagy in many different cell types and experimental conditions (Kim et al, 2007; Carreira et al, 2010; Domenech et al, 2015; Mauro-Lizcano et al, 2015). Inhibition of either autophagy or mitophagy for 6 h prevented the decrease in TOMM20 labelling (Figs 2A and B, and EV1B) and resulted in increased MTDR staining, as measured by flow cytometry (Fig 2C and D). Corroborating these results, immunofluorescence and immunoblot analyses revealed increased expression of the mitochondria respiratory chain component COX-IV (Fig EV1C and D). Incubation of E15.5 retinas with a combination of 3-MA and CsA failed to further increase mitochondrial number compared with either compound alone, suggesting that both inhibitors act on the same signalling pathway (Fig EV1E). Together, these results indicate that the autophagy-dependent elimination of mitochondria during retinal development occurs specifically at E15.5. Figure 2. A mitophagy-dependent metabolic shift towards glycolysis occurs during embryonic retinal development A. Mitochondrial labelling by TOMM20 immunostaining in flat-mounted retinas (E15.5) incubated for 6 h in the presence of 5 μM CsA or 10 mM 3-MA. TOMM20 immunostaining is shown in red and DAPI-stained nuclei in blue. A single confocal plane is shown and corresponds to the inset from the lower magnification images displayed in Fig EV1B. Scale bar, 20 μm. B. Quantification of TOMM20 immunostaining in retinas cultured as in (A) at the indicated embryonic stages (n = 4–16 retinas per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 (Mann-Whitney U-test). C, D. Quantification of the percentage mean fluorescence intensity (% MitoTracker) and representative flow cytometry histograms at the indicated embryonic stages in retinal explants cultured as in (A) and stained with MTDR (n = 6–57 retinas per group). Data are presented as mean ± SEM. *P < 0.05 (Mann-Whitney U-test and Student's t-test). E. Heat map indicating expression of glycolysis-related metabolites in mouse retinas at different developmental stages. P0, Postnatal day 0. F, G. Extracellular acidification rate (ECAR) in embryonic retinas at the indicated developmental stages (F) (n = 19–49 pools of retinas per group) and in E15.5 retinas incubated for 6 h in the presence of 3-MA or CsA (G) (n = 15–19 pools of two retinas per group). Data are presented as mean ± SEM. *P < 0.05 (Mann-Whitney U-test (F) and Student's t-test (G)). H. Heat map showing the relative mRNA expression of glycolysis regulators as determined by transcriptomic analyses in mouse retinas at the indicated developmental stages. I. mRNA expression of glycolysis regulators in E15.5 retinas incubated for 6 h with 3-MA or CsA (n = 3 pools of two retinas per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 (Mann-Whitney U-test). Download figure Download PowerPoint Mitophagy-dependent metabolic reprogramming in retinal development Our previous findings in cell lines suggest that high levels of mitophagy may result in a shift from an oxidative to a glycolytic metabolic profile (Domenech et al, 2015). We next used mass spectrometry to measure the levels of selected metabolites during mouse retinal development. Levels of glycolytic metabolites, including lactate, increased from E15.5, but subsequently decreased at birth (Fig 2E). In agreement, cellular metabolic analyses using Seahorse technology revealed that the extracellular acidification rate (ECAR), which is an indirect measurement of glycolysis, increased at E15.5, but decreased at later developmental stages (Fig 2F). This increase in ECAR was prevented by incubation for 6 h with 3-MA or CsA (Fig 2G). As expected, mitochondrial respiration decreased at E15.5 and increased in retinas treated with 3-MA or CsA (Appendix Fig S3A and B). RNA transcriptome analyses showed a dramatic increase in mRNA expression of multiple genes encoding glycolytic enzymes (Hk2, Pfkfb3 and Gapdh) and glucose transporters (Scla2a1 and Scla2a3) beginning at E15.5 (Fig 2H). Interestingly, treatment of E15.5 retinal explants with 3-MA or CsA significantly reduced mRNA expression of these enzymes, implicating mitophagy in the metabolic shift towards glycolysis during embryonic retinal development (Fig 2I). Mitophagy-dependent metabolic reprogramming and differentiation of macrophages We next investigated whether mitophagy could also play a critical role in other differentiation pathways associated with increased glycolysis. Metabolic reprogramming towards glycolysis has been well described during M1 macrophage activation (O'Neill & Pearce, 2016). Macrophages can be broadly classified into two groups. M1 macrophages, generated in response to proinflammatory conditions such as TLR agonists in combination with IFN-γ, play important roles in the elimination of bacterial infections and are considered more inflammatory (O'Neill & Pearce, 2016). IL-4-activated (M2) macrophages participate in tissue repair and have anti-inflammatory properties (O'Neill & Pearce, 2016). M1 and M2 macrophages also differ in their metabolic signatures. M1 macrophages display a glycolytic profile, characterized by increased expression of several glycolytic regulators and enhanced lactate production, while M2 macrophages show high levels of oxidative phosphorylation (Rodriguez-Prados et al, 2010; Izquierdo et al, 2015; Jha et al, 2015). Crucially, macrophages can be reprogrammed by targeting their metabolism (Izquierdo et al, 2015; Mills & O'Neill, 2016). To explore the potential role of mitophagy in M1 macrophage polarization, we induced the differentiation of peritoneal macrophages into either the M1 or M2 phenotype by incubation with LPS/INF-γ and IL-4/IL-13, respectively (Fig EV2A). M1 macrophages showed decreased MTDR staining as compared with M2 macrophages, indicating a reduction in mitochondrial mass (Fig EV2B). MTDR levels dramatically increased when M1, but not M2, macrophages were cultured in the presence of 3-MA or CsA (Fig EV2C and D). 3-MA and CsA treatment altered cell morphology from the round shape characteristic of the M1 phenotype to the more elongated shape associated with the M2 activation state (Fig EV2E and F). Moreover, mRNA expression of M1 markers such as Tnf, Nos2 and Il1b, as well as expression of several glycolytic enzymes, was reduced when M1 macrophages were incubated in the presence of autophagy or mitophagy inhibitors (Fig EV2G). Together, these data indicate that mitophagy regulates the glycolytic shift associated with cellular differentiation in several cell types. Click here to expand this figure. Figure EV2. Mitophagy sustains glycolysis in M1 macrophages A. Schematic showing experimental design. B. Mouse peritoneal macrophages were isolated from adult mice treated for 4 days with thioglycolate and were then incubated in the presence of LPS and IFN-γ to induce M1 polarization or with IL-4/IL-13 to induce the M2 phenotype. Cells were then stained with MitoTracker and assessed by flow cytometry (n = 12 per group). Data are presented as mean ± SEM. ***P < 0.001 (Mann-Whitney U-test). C, D. M1 (C) or M2 (D) macrophages were incubated for 6 h with 10 mM 3-MA or 5 μM CsA, and MitoTracker staining was assessed by flow cytometry (C, n = 6–9 per group; D, n = 6–9 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 (Mann-Whitney U-test). E, F. Representative images of M1 macrophages (E) after incubation with 3-MA or CsA and M2 macrophages (F). Scale bars, 50 μm. G. mRNA expression of the indicated genes in M1 macrophages cultured in the presence of 10 mM 3-MA or 5 μM CsA (n = 6 per group). Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 (ANOVA). Download figure Download PowerPoint Autophagy-dependent metabolic reprogramming regulates RGC differentiation We next investigated whether mitophagy and the concomitant metabolic shift observed during retinal development are implicated in the neuronal differentiation of RGCs. Inhibition of autophagy or mitophagy with CsA or 3-MA resulted in a decrease in the number of cells positive for the RGC-specific transcription factor Brn3a at E15.5 (Fig 3A and C). Moreover, blockade of autophagy and mitophagy resulted in alterations in axonal morphology as determined by β-III-tubulin labelling in retinal flatmounts (Fig 3B and D), in line with the view that inhibition of autophagy and mitophagy attenuates RGC differentiation. Next, we investigated whether experimental induction of autophagy stimulates RGC differentiation. In the developing retina, neurogenesis follows a central-to-peripheral gradient, meaning that the first RGCs are generated close to the optic nerve at E13.5 (Appendix Fig S1A and B). Pharmacological induction of autophagy with rapamycin at E13.5 increased the num

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. When these tumors are in advanced stages, few therapeutic options are available. Therefore, it is essential to search for new treatments to fight this disease. In this study, we investigated the effects of cannabinoids--a novel family of potential anticancer agents--on the growth of HCC. We found that Δ(9)-tetrahydrocannabinol (Δ(9)-THC, the main active component of Cannabis sativa) and JWH-015 (a cannabinoid receptor 2 (CB(2)) cannabinoid receptor-selective agonist) reduced the viability of the human HCC cell lines HepG2 (human hepatocellular liver carcinoma cell line) and HuH-7 (hepatocellular carcinoma cells), an effect that relied on the stimulation of CB(2) receptor. We also found that Δ(9)-THC- and JWH-015-induced autophagy relies on tribbles homolog 3 (TRB3) upregulation, and subsequent inhibition of the serine-threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation. Pharmacological and genetic inhibition of AMPK upstream kinases supported that calmodulin-activated kinase kinase β was responsible for cannabinoid-induced AMPK activation and autophagy. In vivo studies revealed that Δ(9)-THC and JWH-015 reduced the growth of HCC subcutaneous xenografts, an effect that was not evident when autophagy was genetically of pharmacologically inhibited in those tumors. Moreover, cannabinoids were also able to inhibit tumor growth and ascites in an orthotopic model of HCC xenograft. Our findings may contribute to the design of new therapeutic strategies for the management of HCC.

Cell division is a complex process with high energy demands. However, how cells regulate the generation of energy required for DNA synthesis and chromosome segregation is not well understood. Recent data suggest that changes in mitochondrial dynamics and metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis crosstalk with, and are tightly regulated by, the cell division machinery. Alterations in energy availability trigger cell-cycle checkpoints, suggesting a bidirectional connection between cell division and general metabolism. Some of these connections are altered in human disease, and their manipulation may help in designing therapeutic strategies for specific diseases including cancer. We review here recent studies describing the control of metabolism by the cell-cycle machinery.

Inhibition of a main regulator of cell metabolism, the protein kinase mTOR, induces autophagy and inhibits cell proliferation. However, the molecular pathways involved in the cross-talk between these two mTOR-dependent cell processes are largely unknown. Here we show that the scaffold protein AMBRA1, a member of the autophagy signalling network and a downstream target of mTOR, regulates cell proliferation by facilitating the dephosphorylation and degradation of the proto-oncogene c-Myc. We found that AMBRA1 favours the interaction between c-Myc and its phosphatase PP2A and that, when mTOR is inhibited, it enhances PP2A activity on this specific target, thereby reducing the cell division rate. As expected, such a de-regulation of c-Myc correlates with increased tumorigenesis in AMBRA1-defective systems, thus supporting a role for AMBRA1 as a haploinsufficient tumour suppressor gene.

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in regulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk6(-/-) HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more susceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcriptional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABL(p210+) LSCs. Transplantation with BCR-ABL(p210+)-infected bone marrow from Cdk6(-/-) mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk6(-/-) BCR-ABL(p210+) LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs.

Endocannabinoids act as neuromodulatory and neuroprotective cues by engaging type 1 cannabinoid receptors. These receptors are highly abundant in the basal ganglia and play a pivotal role in the control of motor behaviour. An early downregulation of type 1 cannabinoid receptors has been documented in the basal ganglia of patients with Huntington's disease and animal models. However, the pathophysiological impact of this loss of receptors in Huntington's disease is as yet unknown. Here, we generated a double-mutant mouse model that expresses human mutant huntingtin exon 1 in a type 1 cannabinoid receptor-null background, and found that receptor deletion aggravates the symptoms, neuropathology and molecular pathology of the disease. Moreover, pharmacological administration of the cannabinoid Δ(9)-tetrahydrocannabinol to mice expressing human mutant huntingtin exon 1 exerted a therapeutic effect and ameliorated those parameters. Experiments conducted in striatal cells show that the mutant huntingtin-dependent downregulation of the receptors involves the control of the type 1 cannabinoid receptor gene promoter by repressor element 1 silencing transcription factor and sensitizes cells to excitotoxic damage. We also provide in vitro and in vivo evidence that supports type 1 cannabinoid receptor control of striatal brain-derived neurotrophic factor expression and the decrease in brain-derived neurotrophic factor levels concomitant with type 1 cannabinoid receptor loss, which may contribute significantly to striatal damage in Huntington's disease. Altogether, these results support the notion that downregulation of type 1 cannabinoid receptors is a key pathogenic event in Huntington's disease, and suggest that activation of these receptors in patients with Huntington's disease may attenuate disease progression.

Abstract Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and impairing tumor angiogenesis. It has also been reported that these compounds inhibit tumor cell spreading, but the molecular targets of this cannabinoid action remain elusive. Here, we evaluated the effect of cannabinoids on matrix metalloproteinase (MMP) expression and its effect on tumor cell invasion. Local administration of Δ9-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated MMP-2 expression in gliomas generated in mice, as determined by Western blot, immunofluorescence, and real-time quantitative PCR analyses. This cannabinoid-induced inhibition of MMP-2 expression in gliomas (a) was MMP-2–selective, as levels of other MMP family members were unaffected; (b) was mimicked by JWH-133, a CB2 cannabinoid receptor–selective agonist that is devoid of psychoactive side effects; (c) was abrogated by fumonisin B1, a selective inhibitor of ceramide biosynthesis; and (d) was also evident in two patients with recurrent glioblastoma multiforme. THC inhibited MMP-2 expression and cell invasion in cultured glioma cells. Manipulation of MMP-2 expression by RNA interference and cDNA overexpression experiments proved that down-regulation of this MMP plays a critical role in THC-mediated inhibition of cell invasion. Cannabinoid-induced inhibition of MMP-2 expression and cell invasion was prevented by blocking ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As MMP-2 up-regulation is associated with high progression and poor prognosis of gliomas and many other tumors, MMP-2 down-regulation constitutes a new hallmark of cannabinoid antitumoral activity. [Cancer Res 2008;68(6):1945–52]

GPR55 is an orphan G protein-coupled receptor that may be engaged by some lipid ligands such as lysophosphatidylinositol and cannabinoid-type compounds. Very little is known about its expression pattern and physio-pathological relevance, and its pharmacology and signaling are still rather controversial. Here we analyzed the expression and function of GPR55 in cancer cells. Our data show that GPR55 expression in human tumors from different origins correlates with their aggressiveness. Moreover, GPR55 promotes cancer cell proliferation, both in cell cultures and in xenografted mice, through the overactivation of the extracellular signal-regulated kinase cascade. These findings reveal the importance of GPR55 in human cancer, and suggest that it could constitute a new biomarker and therapeutic target in oncology.

Identifying the molecular mechanisms responsible for the resistance of gliomas to anticancer treatments is an issue of great therapeutic interest. Δ(9)-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the stimulation of autophagy-mediated apoptosis in tumor cells. Here, by analyzing the gene expression profile of a large series of human glioma cells with different sensitivity to cannabinoid action, we have identified a subset of genes specifically associated to THC resistance. One of these genes, namely that encoding the growth factor midkine (Mdk), is directly involved in the resistance of glioma cells to cannabinoid treatment. We also show that Mdk mediates its protective effect via the anaplastic lymphoma kinase (ALK) receptor and that Mdk signaling through ALK interferes with cannabinoid-induced autophagic cell death. Furthermore, in vivo Mdk silencing or ALK pharmacological inhibition sensitizes cannabinod-resistant tumors to THC antitumoral action. Altogether, our findings identify Mdk as a pivotal factor involved in the resistance of glioma cells to THC pro-autophagic and antitumoral action, and suggest that selective targeting of the Mdk/ALK axis could help to improve the efficacy of antitumoral therapies for gliomas.

Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis.

PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.

Abstract Purpose: ABTL0812 is a novel first-in-class, small molecule which showed antiproliferative effect on tumor cells in phenotypic assays. Here we describe the mechanism of action of this antitumor drug, which is currently in clinical development. Experimental Design: We investigated the effect of ABTL0812 on cancer cell death, proliferation, and modulation of intracellular signaling pathways, using human lung (A549) and pancreatic (MiaPaCa-2) cancer cells and tumor xenografts. To identify cellular targets, we performed in silico high-throughput screening comparing ABTL0812 chemical structure against ChEMBL15 database. Results: ABTL0812 inhibited Akt/mTORC1 axis, resulting in impaired cancer cell proliferation and autophagy-mediated cell death. In silico screening led us to identify PPARs, PPARα and PPARγ as the cellular targets of ABTL0812. We showed that ABTL0812 activates both PPAR receptors, resulting in upregulation of Tribbles-3 pseudokinase (TRIB3) gene expression. Upregulated TRIB3 binds cellular Akt, preventing its activation by upstream kinases, resulting in Akt inhibition and suppression of the Akt/mTORC1 axis. Pharmacologic inhibition of PPARα/γ or TRIB3 silencing prevented ABTL0812-induced cell death. ABTL0812 treatment induced Akt inhibition in cancer cells, tumor xenografts, and peripheral blood mononuclear cells from patients enrolled in phase I/Ib first-in-human clinical trial. Conclusions: ABTL0812 has a unique and novel mechanism of action, that defines a new and drugable cellular route that links PPARs to Akt/mTORC1 axis, where TRIB3 pseudokinase plays a central role. Activation of this route (PPARα/γ-TRIB3-Akt-mTORC1) leads to autophagy-mediated cancer cell death. Given the low toxicity and high tolerability of ABTL0812, our results support further development of ABTL0812 as a promising anticancer therapy. Clin Cancer Res; 22(10); 2508–19. ©2015 AACR.

AbstractΔ9-tetrahydrocannabinol (THC), the main active component of marijuana, is being investigated as a potential anti-tumoral agent. We find that THC stimulates an endoplasmic reticulum (ER) stress-related signaling pathway, which activates autophagy via inhibition of the Akt/mTORC1 axis. We also show that autophagy is upstream of apoptosis in cannabinoid-induced cancer cell death and that activation of this pathway is necessary for the anti-tumoral action of cannabinoids in vivo.This article refers to:

Δ9-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer. This effect relies, at least in part, on the up-regulation of several endoplasmic reticulum stress-related proteins including the pseudokinase tribbles homologue-3 (TRIB3), which leads in turn to the inhibition of the AKT/mTORC1 axis and the subsequent stimulation of autophagy-mediated apoptosis in tumor cells. Here, we took advantage of the use of cells derived from Trib3-deficient mice to investigate the precise mechanisms by which TRIB3 regulates the anti-cancer action of THC. Our data show that RasV12/E1A-transformed embryonic fibroblasts derived from Trib3-deficient mice are resistant to THC-induced cell death. We also show that genetic inactivation of this protein abolishes the ability of THC to inhibit the phosphorylation of AKT and several of its downstream targets, including those involved in the regulation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) axis. Our data support the idea that THC-induced TRIB3 up-regulation inhibits AKT phosphorylation by regulating the accessibility of AKT to its upstream activatory kinase (the mammalian target of rapamycin complex 2; mTORC2). Finally, we found that tumors generated by inoculation of Trib3-deficient cells in nude mice are resistant to THC anticancer action. Altogether, the observations presented here strongly support that TRIB3 plays a crucial role on THC anti-neoplastic activity. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Glioblastoma multiforme (GBM) is the most frequent and aggressive form of brain cancer. These features are explained at least in part by the high resistance exhibited by these tumors to current anticancer therapies. Thus, the development of novel therapeutic approaches is urgently needed to improve the survival of the patients suffering this devastating disease. Δ9-Tetrahydrocannabinol (THC, the major active ingredient of marijuana), and other cannabinoids have been shown to exert antitumoral actions in animal models of cancer, including glioma. The mechanism of these anticancer actions relies, at least in part, on the ability of these compounds to stimulate autophagy-mediated apoptosis in tumor cells. Previous observations from our group demonstrated that local administration of THC (or of THC + CBD at a 1:1 ratio, a mixture that resembles the composition of the cannabinoid-based medicine Sativex®) in combination with Temozolomide, the benchmark agent for the treatment of GBM, synergistically reduces the growth of glioma xenografts. With the aim of optimizing the possible clinical utilization of cannabinoids in anti-GBM therapies, in this work we explored the anticancer efficacy of the systemic administration of cannabinoids in combination with TMZ in preclinical models of glioma. Our results show that oral administration of Sativex-like extracts (containing THC and CBD at a 1:1 ratio) in combination with TMZ produces a strong antitumoral effect in both subcutaneous and intracranial glioma cell-derived tumor xenografts. In contrast, combined administration of Sativex-like and BCNU (another alkylating agent used for the treatment of GBM which share structural similarities with the TMZ) did not show a stronger effect than individual treatments. Altogether, our findings support the notion that the combined administration of TMZ and oral cannabinoids could be therapeutically exploited for the management of GBM.

Abstract Fusions transcripts have been proven to be strong drivers for neoplasia-associated mutations, although their incidence in T-cell lymphoblastic lymphoma needs to be determined yet. Using RNA-Seq we have selected 55 fusion transcripts identified by at least two of three detection methods in the same tumour. We confirmed the existence of 24 predicted novel fusions that had not been described in cancer or normal tissues yet, indicating the accuracy of the prediction. Of note, one of them involves the proto oncogene TAL1 . Other confirmed fusions could explain the overexpression of driver genes such as COMMD3-BMI1 , LMO1 or JAK3 . Five fusions found exclusively in tumour samples could be considered pathogenic ( NFYG-TAL1 , RIC3-TCRBC2 , SLC35A3-HIAT1 , PICALM MLLT10 and MLLT10-PICALM ). However, other fusions detected simultaneously in normal and tumour samples ( JAK3-INSL3 , KANSL1-ARL17A/B and TFG-ADGRG7 ) could be germ-line fusions genes involved in tumour-maintaining tasks. Notably, some fusions were confirmed in more tumour samples than predicted, indicating that the detection methods underestimated the real number of existing fusions. Our results highlight the potential of RNA-Seq to identify new cryptic fusions, which could be drivers or tumour-maintaining passenger genes. Such novel findings shed light on the searching for new T-LBL biomarkers in these haematological disorders.

Aim: A 3-month pilot study to evaluate the safety of injecting a bone marrow-derived mesenchymal stem cell extracellular vesicle advanced investigational product (IP) into the lumbar facet joint space as a treatment for chronic low back pain. Methods: 20 healthy adults were treated with IP injections (0.5 ml/joint) and evaluated by three functional assessments 1, 3, 7, 14, 30, 60 and 90 days later. Results: No adverse effects or complications occurred across the 3-month follow-up. There were no reports of worsening pain. After 3 months group average scores improved significantly (p < 0.0001) in the Severity Index (65.04%), Interference Index (72.09%) and Oswestry Disability Index (58.43%) assessments. Conclusion: IP injections were safe and associated with significant functional improvements.What is this article about? Bone marrow mesenchymal stem cell derived extracellular vesicles (BM-MSC EV), a novel biologic therapeutic candidate, are a safe and promising therapeutic intervention for patients with lumbar facet joint pain, a malady that manifests as persistent low back pain (LBP). 20 adult subjects with lumbar facet joint pain received a single injection of BM-MSC EV investigational product in the lumbar facet joint space. What were the results? Follow-up was conducted through in-office and virtual visits that included outcome measures to determine the safety and efficacy of this therapy. By the 3-month end point, follow-up was successful, and no complications or adverse events were noted. Significant improvements in all three assessments of pain and disability occurred throughout the study. What do the results of the study mean? The results are promising and suggest that BM-MSC EV may represent a revolutionary treatment option with durable efficacy and minimal safety risks. Randomized, controlled clinical studies into the application of BM-MSC EV in lumbar facet joint pain should be pursued to confirm the potential benefits of this novel intervention.

Nat. Cell Biol. 17, 20–30 (2015); published online 1 December 2014; corrected after print 1 April 2015 In the version of this Article originally published, incorrect western blot scans were provided for the actin panels in Figure 4h,i. These panels have been corrected online and are shown above. Allsamples in 4i were collected and processed simultaneously, on the same or on parallel gels/blots.

The molecular mechanisms involved in modulation of the antioxidant cell defence by survival signals remain largely unexplored. Here, we report a mechanistic connection between the survival signal elicited by nerve growth factor (NGF) and the antioxidant cell defence represented by heme oxygenase-1 (HO-1) at the level of a newly identified Sp1 site in the human ho1 proximal promoter. By using luciferase reporter constructs we identified a PI3K-responsive region containing a GC-box that resembled the response element for Sp1. Indeed, transfection of Sp1-deficient SL2 cells, electrophoretic mobility shift assays, the use of the GC-box binding drug mithramycin, and mutation of the GC-box provided evidence for a Sp1-like site in the PI3K-sensitive region. Then, we observed with the use of a Sp1–Gal4 chimera that PI3K regulates the transactivating capacity of Sp1. Cotransfection of active PI3K and PKC-ζ expression vectors resulted in substantial increase of Sp1 phosphorylation and in synergistic activation of both Sp1-Gal4 and endogenous Sp1. Moreover, these effects were mimicked by cotransfection of active MEK and ERK expression vectors and were blocked by the MEK inhibitor PD98059. Inhibition of HO-1 with Sn protoporphyrin IX and blockage of Sp-1-mediatied upregulation of HO-1 with mithramycin attenuated antioxidant and cytoprotective functions of NGF against hydrogen peroxide. This study elucidates how NGF contributes to protection of target cells against oxidative stress.

Abstract The aetiology of idiopathic Parkinson's disease (PD) is poorly defined but environmental aggressions may be relevant. Here, we report a new model of PD in mice, based on chronic inoculation with neurotoxins in the nasal cavity, which is a natural route of contact with the environment. C57BL/6 mice, submitted to daily intranasal inoculation with MPTP for 30 days, developed motor deficits that correlated with a progressive and severe depletion of striatal dopamine levels, and loss of tyrosine hydroxylase and dopamine transporter staining in substantia nigra and striatum. Moreover, mice intranasally inoculated with MPTP developed strong astrogliosis and microgliosis in substantia nigra and striatum. Consistent with these observations, a role for oxidant aggression was demonstrated by increased levels of Mn‐superoxide dismutase. However, α‐synuclein aggregation was not observed. This new animal model provides a new tool for studying PD symptoms that develop slowly over time, and it may be used to asses risk from environmental neurotoxins.

Cannabinoids, the active components of Cannabis sativa L. and their derivatives, inhibit tumor growth in laboratory animals by inducing apoptosis of tumor cells and inhibiting tumor angiogenesis. It has also been reported that cannabinoids inhibit tumor cell invasiveness, but the molecular targets of this cannabinoid action remain elusive. Here we evaluated the effects of cannabinoids on the expression of tissue inhibitors of metalloproteinases (TIMPs), which play critical roles in the acquisition of migrating and invasive capacities by tumor cells. Local administration of Δ9-tetrahydrocannabinol (THC), the major active ingredient of cannabis, down-regulated TIMP-1 expression in mice bearing subcutaneous gliomas, as determined by Western blot and immunofluorescence analyses. This cannabinoid-induced inhibition of TIMP-1 expression in gliomas (i) was mimicked by JWH-133, a selective CB2 cannabinoid receptor agonist that is devoid of psychoactive side effects, (ii) was abrogated by fumonisin B1, a selective inhibitor of ceramide synthesis de novo, and (iii) was also evident in two patients with recurrent glioblastoma multiforme (grade IV astrocytoma). THC also depressed TIMP-1 expression in cultures of various human glioma cell lines as well as in primary tumor cells obtained from a glioblastoma multiforme patient. This action was prevented by pharmacological blockade of ceramide biosynthesis and by knocking-down the expression of the stress protein p8. As TIMP-1 up-regulation is associated with high malignancy and negative prognosis of numerous cancers, TIMP-1 down-regulation may be a hallmark of cannabinoid-induced inhibition of glioma progression.

Abstract Gene expression profiling has revealed that the gene coding for cannabinoid receptor 1 (CB1) is highly up-regulated in rhabdomyosarcoma biopsies bearing the typical chromosomal translocations PAX3/FKHR or PAX7/FKHR. Because cannabinoid receptor agonists are capable of reducing proliferation and inducing apoptosis in diverse cancer cells such as glioma, breast cancer, and melanoma, we evaluated whether CB1 is a potential drug target in rhabdomyosarcoma. Our study shows that treatment with the cannabinoid receptor agonists HU210 and Δ9-tetrahydrocannabinol lowers the viability of translocation-positive rhabdomyosarcoma cells through the induction of apoptosis. This effect relies on inhibition of AKT signaling and induction of the stress-associated transcription factor p8 because small interfering RNA–mediated down-regulation of p8 rescued cell viability upon cannabinoid treatment. Finally, treatment of xenografts with HU210 led to a significant suppression of tumor growth in vivo. These results support the notion that cannabinoid receptor agonists could represent a novel targeted approach for treatment of translocation-positive rhabdomyosarcoma. [Mol Cancer Ther 2009;8(7):1838–45]

Abstract Gliomas, one of the most malignant forms of cancer, exhibit high resistance to conventional therapies. Identification of the molecular mechanisms responsible for this resistance is therefore of great interest to improve the efficacy of the treatments against these tumors. Δ9‐Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer, including glioma, an effect that relies, at least in part, on the ability of these compounds to induce apoptosis of tumor cells. By analyzing the gene expression profile of two sub‐clones of C6 glioma cells with different sensitivity to cannabinoid‐induced apoptosis, we found a subset of genes with a marked differential expression in the two sub‐clones. Furthermore, we identified the epidermal growth factor receptor ligand amphiregulin as a candidate factor to mediate the resistance of glioma cells to cannabinoid treatment. Amphiregulin was highly overexpressed in the cannabinoid‐resistant cell line, both in culture and in tumor xenografts. Moreover, in vivo silencing of amphiregulin rendered the resistant tumors xenografts sensitive to cannabinoid antitumoral action. Amphiregulin expression was associated with increased extracellular signal‐regulated kinase (ERK) activation, which mediated the resistance to THC by blunting the expression of p8 and TRB3—two genes involved in cannabinoid‐induced apoptosis of glioma cells. Our findings therefore identify Amphirregulin as a factor for resistance of glioma cells to THC‐induced apoptosis and contribute to unraveling the molecular bases underlying the emerging notion that targeted inhibition of the EGFR pathway can improve the efficacy of antitumoral therapies. © 2009 Wiley‐Liss, Inc.

Abstract Spatio-temporal regulation of intracellular signalling networks is key to normal cellular physiology; dysregulation of which leads to disease. The family of three mammalian tribbles proteins has emerged as an important controller of signalling via regulating the activity of mitogen activated protein kinases (MAPK), the PI3-kinase induced signalling network and E3 ubiquitin ligases. However, the importance of potential redundancy in the action of tribbles and how the differences in affinities for the various binding partners may influence signalling control is currently unclear. We report that tribbles proteins can bind to an overlapping set of MAPK-kinases (MAPKK) in live cells and dictate the localisation of the complexes. Binding studies in transfected cells reveal common regulatory mechanisms and suggest that tribbles and MAPKs may interact with MAPKKs in a competitive manner. Computational modelling of the impact of tribbles on MAPK activation suggests a high sensitivity of this system to changes in tribbles levels, highlighting that these proteins are ideally placed to control the dynamics and balance of activation of concurrent signalling pathways.

Abstract Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin‐dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK‐counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline‐directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome‐dependent degradation. Multiomic single‐cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK‐CDC14 axis as a critical molecular switch for linking cell cycle regulation and self‐renewal.

Δ9-tetrahydrocannabinol (THC), the main active component of marijuana, promotes cancer cell death via autophagy stimulation. We find that activation of the tyrosine kinase receptor ALK by its ligand midkine interferes with the signaling mechanism by which THC promotes autophagy-mediated glioma cell death.

Different physiological and pathological situations that produce alterations in the endoplasmic reticulum, lead to a condition known as ER stress. ER stress activates a complex intracellular signal transduction pathway, called unfolded protein response (UPR). UPR is tailored essentially to reestablish ER homeostasis. However, when persistent, ER stress can switch the cytoprotective functions of UPR into cell death promoting mechanisms. One of the cellular mechanisms that are regulated by ER stress is autophagy. Autophagy is a cellular process by which different cytoplasmic components including organelles are targeted for degradation to the autophagosomes. Interestingly, like ER stress, autophagy can be a protective or a cell death promoting mechanism. Recently, a variety of anticancer therapies (including those that stimulate ER stress) have been shown to activate autophagy in tumor cells, which has been proposed to either enhance cancer cell death or act as a mechanism of resistance to chemotherapy. In this chapter, we will describe some of the procedures that are currently used to analyze autophagy as well as some of the experimental approaches that can be undertaken to investigate the connection between ER stress and autophagy in cancer.

Tribbles pseudokinase 3 (TRIB3) belongs to the tribbles family of pseudokinases. In this article, we summarize several observation obtained by our laboratories supporting that TRIB3 plays a crucial role in the anti-cancer activity of cannabinoids (a novel family of potential anti-cancer agents derived from marijuana) and that TRIB3 genetic inactivation enhances cancer generation and progression.

Article2 July 2020free access Source DataTransparent process Transient exposure to miR-203 enhances the differentiation capacity of established pluripotent stem cells María Salazar-Roa Corresponding Author [email protected] orcid.org/0000-0001-6784-9541 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marianna Trakala Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Mónica Álvarez-Fernández Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Fátima Valdés-Mora orcid.org/0000-0001-7490-0114 Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia St. Vincent's Clinical School, UNSW, Sydney, Sydney, NSW, Australia Search for more papers by this author Cuiqing Zhong Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Jaime Muñoz Transgenics Unit, CNIO, Madrid, Spain Search for more papers by this author Yang Yu Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Timothy J Peters Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Osvaldo Graña-Castro Bioinformatics Unit, CNIO, Madrid, Spain Search for more papers by this author Rosa Serrano Telomeres and Telomerase Group, CNIO, Madrid, Spain Search for more papers by this author Elisabet Zapatero-Solana Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author María Abad Tumor Suppression Group, CNIO, Madrid, Spain Search for more papers by this author María José Bueno Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marta Gómez de Cedrón Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author José Fernández-Piqueras orcid.org/0000-0003-4520-6785 Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red para Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain Instituto de Investigación Biosanitaria, Fundación Jimenez Díaz, Madrid, Spain Search for more papers by this author Manuel Serrano orcid.org/0000-0001-7177-9312 Tumor Suppression Group, CNIO, Madrid, Spain Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author María A Blasco orcid.org/0000-0002-4211-233X Telomeres and Telomerase Group, CNIO, Madrid, Spain Search for more papers by this author Da-Zhi Wang Cardiovascular Research Division, Boston Children′s Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Susan J Clark Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia St. Vincent's Clinical School, UNSW, Sydney, Sydney, NSW, Australia Search for more papers by this author Juan Carlos Izpisua-Belmonte Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Sagrario Ortega Transgenics Unit, CNIO, Madrid, Spain Search for more papers by this author Marcos Malumbres Corresponding Author [email protected] orcid.org/0000-0002-0829-6315 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author María Salazar-Roa Corresponding Author [email protected] orcid.org/0000-0001-6784-9541 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marianna Trakala Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Mónica Álvarez-Fernández Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Fátima Valdés-Mora orcid.org/0000-0001-7490-0114 Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia St. Vincent's Clinical School, UNSW, Sydney, Sydney, NSW, Australia Search for more papers by this author Cuiqing Zhong Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Jaime Muñoz Transgenics Unit, CNIO, Madrid, Spain Search for more papers by this author Yang Yu Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Timothy J Peters Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia Search for more papers by this author Osvaldo Graña-Castro Bioinformatics Unit, CNIO, Madrid, Spain Search for more papers by this author Rosa Serrano Telomeres and Telomerase Group, CNIO, Madrid, Spain Search for more papers by this author Elisabet Zapatero-Solana Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author María Abad Tumor Suppression Group, CNIO, Madrid, Spain Search for more papers by this author María José Bueno Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Marta Gómez de Cedrón Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author José Fernández-Piqueras orcid.org/0000-0003-4520-6785 Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain Centro de Investigación Biomédica en Red para Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain Instituto de Investigación Biosanitaria, Fundación Jimenez Díaz, Madrid, Spain Search for more papers by this author Manuel Serrano orcid.org/0000-0001-7177-9312 Tumor Suppression Group, CNIO, Madrid, Spain Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain Search for more papers by this author María A Blasco orcid.org/0000-0002-4211-233X Telomeres and Telomerase Group, CNIO, Madrid, Spain Search for more papers by this author Da-Zhi Wang Cardiovascular Research Division, Boston Children′s Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Susan J Clark Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia St. Vincent's Clinical School, UNSW, Sydney, Sydney, NSW, Australia Search for more papers by this author Juan Carlos Izpisua-Belmonte Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA Search for more papers by this author Sagrario Ortega Transgenics Unit, CNIO, Madrid, Spain Search for more papers by this author Marcos Malumbres Corresponding Author [email protected] orcid.org/0000-0002-0829-6315 Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain Search for more papers by this author Author Information María Salazar-Roa *,1,‡, Marianna Trakala1,‡, Mónica Álvarez-Fernández1,‡, Fátima Valdés-Mora2,3,‡, Cuiqing Zhong4,‡, Jaime Muñoz5, Yang Yu4, Timothy J Peters2, Osvaldo Graña-Castro6, Rosa Serrano7, Elisabet Zapatero-Solana1, María Abad8, María José Bueno1, Marta Gómez Cedrón1, José Fernández-Piqueras9,10,11, Manuel Serrano8,12,13, María A Blasco7, Da-Zhi Wang14, Susan J Clark2,3, Juan Carlos Izpisua-Belmonte4, Sagrario Ortega5 and Marcos Malumbres *,1 1Cell Division and Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain 2Epigenetics Research Program, Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, NSW, Australia 3St. Vincent's Clinical School, UNSW, Sydney, Sydney, NSW, Australia 4Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, USA 5Transgenics Unit, CNIO, Madrid, Spain 6Bioinformatics Unit, CNIO, Madrid, Spain 7Telomeres and Telomerase Group, CNIO, Madrid, Spain 8Tumor Suppression Group, CNIO, Madrid, Spain 9Centro de Biología Molecular Severo Ochoa (CBMSO), Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid (CSIC-UAM), Madrid, Spain 10Centro de Investigación Biomédica en Red para Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Madrid, Spain 11Instituto de Investigación Biosanitaria, Fundación Jimenez Díaz, Madrid, Spain 12Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 13Catalan Institution for Research and Advanced Studies (ICREA), Barcelona, Spain 14Cardiovascular Research Division, Boston Children′s Hospital, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +34 917 328 000; ext 3452; E-mail: [email protected] *Corresponding author. Tel: +34 917 328 000; ext 3450; E-mail: [email protected] EMBO J (2020)39:e104324https://doi.org/10.15252/embj.2019104324 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Full differentiation potential along with self-renewal capacity is a major property of pluripotent stem cells (PSCs). However, the differentiation capacity frequently decreases during expansion of PSCs in vitro. We show here that transient exposure to a single microRNA, expressed at early stages during normal development, improves the differentiation capacity of already-established murine and human PSCs. Short exposure to miR-203 in PSCs (miPSCs) induces a transient expression of 2C markers that later results in expanded differentiation potency to multiple lineages, as well as improved efficiency in tetraploid complementation and human–mouse interspecies chimerism assays. Mechanistically, these effects are at least partially mediated by direct repression of de novo DNA methyltransferases Dnmt3a and Dnmt3b, leading to transient and reversible erasure of DNA methylation. These data support the use of transient exposure to miR-203 as a versatile method to reset the epigenetic memory in PSCs, and improve their effectiveness in regenerative medicine. Synopsis Long-term expansion of pluripotent stem cells (PSC) frequently results in decreased differentiation potential diminishing their utility. Here, a miRNA-based strategy is shown to enhance PSC potency via resetting their epigenetic state, suggesting new opportunities for improved stem cell therapies. Developmental miR-203 improves potential and plasticity of mouse and human PSCs in vivo. miR-203 increases human-mouse interspecies chimera competency. miR-203 targets de novo DNA methyltransferases Dnmt3a/b inducing global and reversible DNA hypomethylation. Transient miR-203 enhances PSC maturation into cardiomyocytes. Introduction Pluripotent stem cells (PSCs) provide an important promise for regenerative medicine due to their self-renewal potential and ability to differentiate into multiple cell lineages. During the last years, multiple efforts have been put into improving the potential of these cells either by expanding their differentiation capacity into a wider variety of cell lineages or by improving maturation properties into specific functional cell types (Li & Izpisua Belmonte, 2016; Takahashi & Yamanaka, 2016). PSCs are commonly cultured in the presence of Mek1/2 and Gsk3 inhibitors together with the cytokine Lif (2i/L conditions Ying et al, 2008). Although these conditions may improve the maintenance of pluripotency in vitro, recent evidences suggest that prolonged inhibition of Mek1/2 may limit the developmental and differentiation capacity of PSCs in vivo, in part by inducing irreversible demethylation of imprinting control regions (ICRs) (Choi et al, 2017a; Yagi et al, 2017). Recent data suggest that genetic ablation of miR-34 in PSCs results in improved potential to form embryonic and extraembryonic tissues in part by promoting Gata2 expression (Choi et al, 2017b), although the conditions for applying this information to favor the differentiation potential of PSCs remain to be established. Finally, a recent alternative proposal suggests the use of a chemical cocktail of inhibitors that also enhances the developmental potential of PSCs (Yang et al, 2017); however, the applicability of this method is still limited due to the lack of mechanistic details. Here, we identified miR-203 as a microRNA preferentially expressed in the 2C-morula stages during pre-implantation development in the mouse embryo. By using a variety of in vitro and in vivo approaches, we report that transient exposure of already-established induced pluripotent stem cells (iPSCs) or ESCs to miR-203 enhances the ability of these PSCs to differentiate into multiple cell lineages and to reach further maturation properties. Transient expression of miR-203 in PSCs (miPSCs) leads to greater developmental potential in tetraploid complementation assays, and significantly improves the efficiency of human iPSCs in contributing to interspecies human–mouse conceptuses. Mechanistically, these effects are at least partially mediated through the miR-203-dependent control of de novo DNA methyltransferases Dnmt3a and Dnmt3b, thereby regulating the DNA methylation landscape of these miPSCs. These observations suggest that the developmental and differentiation potential of already-established PSCs can be readily enhanced by transient exposure to a single microRNA. Results miR-203 improves the differentiation potency of PSCs miR-203 was originally proposed to limit the stemness potential of skin progenitors (Yi et al, 2008) and to display tumor-suppressive functions in multiple cancers (Bueno et al, 2008; Michel & Malumbres, 2013), suggesting a role in the balance between stemness and differentiation. However, its expression during early development remained undefined. A first analysis of miR-203 levels during normal murine and bovine development suggested a modest but specific wave of expression during early development (blastocyst stage in murine and more particularly hatched blastocysts in bovine embryos), whereas its expression was lost in cultured embryonic stem cells (Yang et al, 2008; Goossens et al, 2013). Interestingly, our quantitative PCR analysis in mouse embryos isolated at different early developmental stages showed that miR-203 expression was low in oocytes, slightly induced at the 2-cell stage, and displayed a gradual reduction in morulas and blastocysts (Fig 1A). Figure 1. Effects of transient induction of miR-203 in iPSC and ESC pluripotency and differentiation potential miR-203 expression, as determined by qPCR, in five temporal different stages of normal early development: oocyte, 2-cell embryo, morula, compacted morula, and blastocyst. RNA was extracted from 30 different embryos and pooled in two independent groups for analysis by qPCR. RNA expression is normalized by a housekeeping miRNA (miR-16) that maintained invariable during early embryogenesis. Data represent the mean of 6 different qPCR measures (red bars). P = 0.05 (Student's t-test) comparing 2C/morula versus compacted morula/blastocyst. Protocol for reprogramming of miR-203 mutant MEFs into pluripotent iPSCs and subsequent differentiation into embryoid bodies. MEFs were transduced with lentiviruses expressing Oct4, Sox2, Klf4, and cMyc (OSKM) in a constitutive manner. The resulting iPSCs were then treated with doxycycline (Dox) 1 μg/ml during 5 days to induce miR-203 expression. “miiPSCs” refers to iPSCs in which miR-203 was transiently expressed during the indicated 5 days. Dox was removed for 15–30 days before starting the embryoid body generation protocol. Samples for RNA sequencing were taken 30 days after Dox withdrawal. Principal component analysis of RNAseq data from wild-type iPSCs (n = 3 clones), miiPSCs (n = 4), and wild-type ESCs (n = 3). Enrichment plots of the 282-gene 2-cell signature (Biase et al, 2014) in miiPSCs 10 and 25 days after Dox withdrawal. PCA of RNAseq and microarray data from miiPSCs and known already-published PSC types. Log2 expression values were normalized to mESCs in each study. Data from miiPSCs (this study), mES cells (each study), 2C-like cells (MacFarlan et al, 2012), and epiblast stem cells (Najm et al, 2011) were analyzed, and a total of 17,243 genes were selected. For miiPSCs, clones 1 and 2 were analyzed 10 days after miR-203 exposure, while clones 3 and 4 were analyzed 25 days after miR-203 exposure (see schematic shown in Fig 1B). For Tomato PSCs, Tomato fluorescence is associated with 2C-like retrotransposon expression, and thus, Tomato-positive cells are identified as 2C-like cells (Macfarlan et al, 2012). Representative images of embryoid bodies (EBs) derived from wild-type iPSCs or ESCs, or from miiPSC and miESCs at day 30 of differentiation. Scale bars, 500 μm. Quantification of the percentage of EBs from panel (F) presenting internal large cavities and EBs beating during the indicated time course. Data are represented as mean ± SEM (n = 3 independent experiments). *P < 0.05; ***P < 0.001 (Student's t-test). Representative images of EBs derived from human iPSCs transiently transfected with either control (left) or miR-203 mimics (right), at different time points during the differentiation process. Scale bars, 500 μm. Left panel shows the quantification of EBs size derived from human iPSCs transiently transfected with either control or miR-203 mimics as in panel (H) at different time points during the differentiation process. The percentage of EBs presenting internal large cavities during the indicated time course of differentiation is shown in the right panel. Data are mean ± SEM (n = 3 independent experiments). ***P < 0.001 (Student's t-test). Source data are available online for this figure. Source Data for Figure 1 [embj2019104324-sup-0010-SDataFig1.xlsx] Download figure Download PowerPoint To easily manipulating miR-203 levels in vitro and in vivo, we generated a tetracycline-inducible knock-in model in which the miR-203-encoding sequence was inserted downstream of the type I collagen gene and expressed under the control of a tetracycline-responsive element [ColA1(miR-203) allele] in the presence of tetracycline reverse transactivator expressed from the Rosa26 locus [Rosa26(rtTA) allele] (Fig EV1A). Treatment of ColA1(miR-203/miR-203); Rosa26(rtTA/rtTA) mouse embryonic fibroblasts (MEFs) or ESCs with doxycycline (Dox) led to a significant induction of miR-203 levels (Fig EV1B). We also generated iPSCs from wild-type or un-induced ColA1(miR-203/miR-203); Rosa26(rtTA/rtTA) MEFs by using lentiviral vectors expressing Oct4, Sox2, Klf4, and cMyc (Takahashi & Yamanaka, 2006). Similar to MEFs or ESCs, these mutant iPSCs showed a significant induction of miR-203 after treatment with doxycycline (Fig EV1B). Although previous reports have suggested that this system suffers of relative leakiness (Stadtfeld et al, 2010b), miR-203 expression significantly reduced and was undetectable a few days after Dox withdrawal (Fig EV1C). Click here to expand this figure. Figure EV1. Mouse alleles generated and used in this work, transcriptomic analysis of miiPSCs, and validation of miR-203 mimics and vectors on EBs differentiation Schematic representation of the miR-203-inducible knock-in model generated in this work. In the ColA1(miR-203/miR-203); Rosa26(rtTA/rtTA) model, the reverse tetracycline transactivator is expressed from the Rosa26 locus, whereas miR-203 is driven by the tetracycline operator downstream of the ColA1 locus. miR-203 expression, as determined by quantitative PCR, in ColA1(miR-203/miR-203); Rosa26(rtTA/rtTA) MEFs, iPSCs, and ESCs treated or not with doxycycline (Dox). miR-203 expression is normalized by a control miRNA (miR-142). Data are mean ± SD (n = 3 independent experiments). ***P < 0.001 (Student's t-test). Time course of miR-203 expression, as determined by qPCR. RNA expression is normalized by a housekeeping miRNA (miR-16) that maintained invariable. Data are mean ± SD (n = 4 independent replicates). P < 0.001 (Student's t-test) comparing Dox treatment (green box) versus Dox withdrawal. Unbiased clustering of genome-wide RNAseq data (left) and heat map plot showing the comparative expression of 450 genes associated with pluripotency (Chung et al, 2012); 2 clones per sample. Early expression (RPKM) of the indicated transcripts included in the 2C signature. Data are mean ± SEM (n = 3 independent experiments). *P < 0.05; **P < 0.01 (Student's t-test). Top categories in the Gene Ontology analysis of the genes significantly upregulated in miiPSCs versus un-induced iPSCs (4 independent clones were analyzed; see also Dataset EV1). P-values were calculated by Fisher's exact test. (Upper panels) Representative images of EBs derived from either wild-type iPSCs (left) or ESCs (right) transduced with empty pMCSV vector, pMCSV-miR-203, or transfected with control mimics or miR-203 mimics, at day 30 of the differentiation process. Scale bars, 500 μm. (Lower panels) Quantification of EBs with large cavities and beating EBs during the differentiation process. Data are mean ± SEM (n = 3 independent experiments). **P < 0.01 (in both iPS and ES cells; Student's t-test). Source data are available online for this figure. Download figure Download PowerPoint We next studied the long-term effects of miR-203 by treating ColA1(miR-203/miR-203); Rosa26(rtTA/rtTA) iPSCs (generated in the absence of Dox) transiently with Dox for 5 days (microRNA transiently induced iPSCs or miiPSCs; Fig 1B). RNA sequencing of these iPSC clones as well as wild-type ESCs was analyzed 10 days or 1 month after miR-203 induction (Fig 1B) and surprisingly revealed that miiPSCs were transcriptionally closer to ESCs than Dox-treated wild-type iPSCs both at the genome-wide level (Fig 1C) and when considering a pluripotency-associated signature defined previously (Chung et al, 2012) (Fig EV1D). In addition, almost every transcript included in a 282-gene signature of 2C blastomeres (Biase et al, 2014) was induced by miR-203 and sustained during 10 days after Dox withdrawal (Figs 1D and EV1E), including genes such as Zscan4c previously related to the zygotic transcriptional program (Eckersley-Maslin et al, 2019). Importantly, we also assessed the transcriptomes of different clones of miiPSCs, mESCs, 2-cell-like mESC subpopulations (MacFarlan et al, 2012; GSE 33923), and epiblast stem cells (Najm et al, 2011; GSE 26814) (Fig 1B and E). Principal component analysis revealed a gene expression pattern of miiPSC clones 1 and 2 (analyzed 10 days after miR-203 exposure) similar to that observed for Tomato-positive cells, characterized as 2C-like ESCs by MacFarlan and collaborators, while miiPSC clones 3 and 4 (analyzed 25 days after miR-203 exposure) exhibited a profile more similar to ESC clones, corroborating the observations showed in Fig 1D. An additional comparison of the expression profiles in miiPSCs versus un-induced iPSCs at day 30 after doxycycline withdrawal suggested higher expression of genes related to tissue morphogenesis and embryonic development in those clones in which miR-203 had been transiently induced (Figs 1B and EV1F and Dataset EV1). The differentiation potential of miiPSCs was directly tested in the embryoid body (EB) formation assay in vitro. Transient induction of miR-203 for 5 days followed by 2–4 weeks of Dox withdrawal in either iPSCs (miiPSCs) or ESCs (miESCs) resulted in a significant increase in EB size accompanied by the formation of large internal cavities (Fig 1F and G, Appendix Fig S1A–C). Furthermore, miiPSC- and miESC-derived EBs showed beating with higher efficiency and at earlier time points than their untreated counterparts (Fig 1G, Appendix Fig S1C). miiPSC-derived EBs displayed a complex organization with high expression of primitive endoderm (Gata4), mesoderm (Cd34), and ectoderm (Pax6) or neuroectoderm (Nestin) markers (Appendix Fig S1D and E and Movie EV1). To further validate these observations beyond the genetic model, we tested the effect of transient expression of ectopic miR-203 using a CMV-driven retroviral vector or RNA mimics (Fig EV1G and Appendix Fig S1F and G). These two strategies also resulted in improved EB formation (large cavities, beating) in miiPSCs or miESCs (Fig EV1G), indicating that these effects were not unique to the inducible genetic model. Since the combination of the MEK inhibitor PD0325901 and the GSK3 inhibitor CHIR99021 with LIF (2i/L conditions) has been previously shown to render iPSCs closer to ESCs (Ying et al, 2008), we also tested the effect of miR-203 under 2i/L conditions. miiPSCs grown in 2i/L also displayed an enrichment in the transcription of stemness factors and developmental pathways when compared to wild-type iPSCs grown in the same conditions (Appendix Fig S2A). In addition, pre-treatment with Dox for 5 days, 2–4 weeks prior to EB assays, of miiPSCs cultured in 2i/L conditions promoted a significant increase in EB size, formation of large internal cavities, and beating (Appendix Fig S2B and C), suggesting an additional effect of miR-203 over the 2i/L conditions. The expression of miR-203 at the 2-cell stage inspired us to further analyze the early expression of 2C-related endogenous retroviruses in cultured miPSCs. Transient expression of miR-203-GFP in wild-type ES cells significantly increased the number of 2C-like cells as determined by the expression of the murine endogenous retrovirus with leucine tRNA primer (MuERV-L; 2C::Td-Tomato reporter; MacFarlan et al, 2012) analyzed 24 h after miR-203 addition (Appendix Fig S3A). In line with these observations, exposure to miR-203 induced the primary expression of genes harboring a proximal upstream or an intronic MERVL element (Appendix Fig S3B). Of interest, transient exposure to miR-203 mimics also rendered human pluripotent cells to a ground naive state (Appendix Fig S3C and D), as measured by the expression of the family HERVH of human endogenous retroviruses (HERVs) involved in the maintenance of human naive pluripotency (Wang & Gao, 2016). miR-203 mimics induced the expression of the HERVH-GFP reporter in a significant number of colonies, in many cases not only in the periphery but also in almost the totality of the cells of the colony, and such expression was sustained for 5 days in culture. When the differentiation potential of these cultures was tested (several weeks after miR-203 transient expression), human miiPSCs generated significantly bigger EBs with larger internal cavities than the control counterparts (Fig 1H and I), suggesting altogether that the effect of miR-203 can be achieved using delivery systems with independence of the genetic knock-in system and it is also functional in human cells. miR-203 expands developmental potential and plasticity of PSCs We then tested the potential of miiPSCs (in which the expression of miR-203 had only been induced for 5 days in vitro) to form teratomas either after subcutaneous or intraperitoneal injection. miiPSCs formed significantly bigger tumors in these assays (Fig 2A). Intriguingly, these teratomas were not only bigger but also they contained tissues that are not typically found in control iPSC-induced teratomas, such as bone marrow, pancreas, or cartilage, as well as trophoblast giant cells as confirmed by the expression of PL-1 (Figs 2B and EV2). Transcriptomic studies in these complex structures suggested upregulation of genes involved in embryonic development and organ morphogenesis when derived from miiPSCs (Appendix Fig S4A and Appendix Table S1). Immunohistochemistry studies showed elevated expression of multiple differentiation markers representing ectoderm, mesoderm, and endoderm in miiPSC-derived teratomas (Appendix Fig S4B and C). Interestingly, these teratomas also showed elevated levels of proliferation (as scored by Ki67; Appendix Fig S4D) or pluripotency markers such as Nanog, Oct4, or Sox2 in vivo (Appendix Fig S4B), suggesting that these structures contained a complex mixture of undifferentiated cells, expressing stemness markers, together with differentiated cells. Figure 2. Transient exposure to miR-203 in vitro improves the in vivo developmental potential of iPSCs and ESCs Teratoma volume (mm3) 20–25 days after subcutaneous injection of wild-type iPSCs or miiPSCs expressing GFP. Data are represented as mean ± SEM (n = 8 tumors per genotype; 292.8 ± 66.67 versus 1,216 ± 140.1; difference between means: 923.5 ± 155.2; 95% confidence interval: 1,256 to −590.7); ***P < 0.001 (Student's t-test). Representative images are show

In a recent article, we found that Tribbles pseudokinase 3 (TRIB3) plays a tumor suppressor role and that this effect relies on the dysregulation of the phosphorylation of v-akt murine thymoma viral oncogene homolog (AKT) by the mammalian target of rapamycin complex 2 (mTORC2 complex), and the subsequent hyperphosphorylation and inactivation of the transcription factor Forkhead box O3 (FOXO3).

Copper (Cu) dyshomeostasis has been linked to obesity and related morbidities and also to aging. Cu levels are higher in older or obese individuals, and adipose tissue (AT) Cu levels correlate with body mass index. Aging and obesity induce similar AT functional and structural changes, including an accumulation of senescent cells. To study the effect of Cu-mediated stress-induced premature senescent (Cu-SIPS) on preadipocytes, 3T3-L1 cell line was exposed to a subcytotoxic concentration of copper sulfate. After Cu treatment, preadipocytes acquired typical senescence characteristics including diminished cell proliferation, cell and nuclei enlargement and increased lysosomal mass (higher Lamp2 expression and a slight increased number of cells positive for β-galactosidase associated with senescence (SA-β-Gal)). Cell cycle arrest was due to upregulation of p16Ink4aInk4a and p21Waf1/Cip1. Accordingly, protein levels of the proliferation marker KI67 were reduced. Cu-SIPS relates with oxidative stress and, in this context, an increase of SOD1 and HO-1 expression was detected in Cu-treated cells. The mRNA expression of senescence-associated secretory phenotype factors, such as Mmp3, Il-6 and Tnf-α, increased in Cu-SIPS 3T3-L1 cells but no effect was observed on the expression of heterochromatin-associated protein 1(HP1). Although the downregulation of LaminB1 expression is considered a hallmark of senescence, Cu-SIPS cells presented higher levels of LaminB1. The dysregulation of nuclear lamina was accompanied by an increase of nuclear blebbing, but not of micronuclei number. To conclude, a Cu-SIPS model in 3T3-L1 preadipocytes is here described, which may be an asset to the study of AT dysregulation observed in obesity and aging.

Polyploidization in megakaryocytes is achieved by endomitosis, a specialized cell cycle in which DNA replication is followed by aberrant mitosis. Typical mitotic regulators such as Aurora kinases or Cdk1 are dispensable for megakaryocyte maturation, and inhibition of mitotic kinases may in fact promote megakaryocyte maturation. However, we show here that Polo-like kinase 1 (Plk1) is required for endomitosis, and ablation of the Plk1 gene in megakaryocytes results in defective polyploidization accompanied by mitotic arrest and cell death. Lack of Plk1 results in defective centrosome maturation and aberrant spindle pole formation, thus impairing the formation of multiple poles typically found in megakaryocytes. In these conditions, megakaryocytes arrest for a long time in mitosis and frequently die. Mitotic arrest in wild-type megakaryocytes treated with Plk1 inhibitors or Plk1-null cells is triggered by the spindle assembly checkpoint (SAC), and can be rescued in the presence of SAC inhibitors. These data suggest that, despite the dispensability of proper chromosome segregation in megakaryocytes, an endomitotic SAC is activated in these cells upon Plk1 inhibition. SAC activation results in defective maturation of megakaryocytes and cell death, thus raising a note of caution in the use of Plk1 inhibitors in therapeutic strategies based on polyploidization regulators.

People diagnosed with severe and persistent mental illness (SPMI) face multiple vulnerabilities, including when seeking employment. Among SPMI patients, studies show that a stronger sense of spirituality can help to reduce psychotic symptoms, increase social integration, reduce the risk of suicide attempts and promote adherence to psychiatric treatment. This study examined how the variables spirituality and employment affect the recovery process and psychological well-being of people with SPMI who attend employment recovery services. The sample consisted of 64 women and men diagnosed with an SPMI. The assessment instruments included the Recovery Assessment Scale, Ryff Psychological Well-Being Scale, Work Motivation Questionnaire, Daily Spiritual Experience Scale, and Functional Assessment of Chronic Illness Therapy—Spiritual Well-Being (FACIT-Sp12). Hierarchical regression analyses were performed to compare three different models for each dependent variable (recovery and psychological well-being). The findings showed that job skills predicted psychological well-being and recovery. When spiritual variables were included in the model, job skills dropped out and the dimension meaning/peace of the FACIT-Sp12 emerged as the only significant predictor variable. Integrating spirituality into recovery programs for people with SPMI may be a helpful complement to facilitate the recovery process and improve psychological well-being.

In this study we intend to understand the impact of the COVID-19 crisis and the subsequent stay-at-home orders, on the Spanish population's sense of belonging at three moments in time: at the beginning of the lockdown, after one month of lockdown and with the return to the "new normality". A cross-sectional study was conducted through an online survey (N0 = 3480; N1 = 1041; N2 = 569). The sense of belonging was evaluated by means of four Likert-type items. These questions included membership in different groups: work/studies, friends, family and neighborhood or community. Sociodemographic and COVID-19-related data were collected. Additionally, mental health, spiritual well-being, loneliness, social support and discrimination were assessed. Descriptive analyses were carried out and linear regression models compiled. The sense of belonging increased significantly during confinement, dropping dramatically with the start of the return to the "new normality" process. The only variable that showed interaction with time and sense of belonging was discrimination. Work condition (not working providing the lowest sense of belonging scores), social support from friends and loneliness were the main predictors of the sense of belonging. The impact caused by the pandemic and the actions adopted during the first weeks regarding the sense of belonging is evident. It has been a key variable in dealing with COVID-19. Actions are now needed to increase our sense of belonging to face the post-epidemic crisis and avoid a greater impact in other areas.

Highly purified cannabidiol (CBD) has a broad spectrum of action and could be useful for the treatment of drug resistant epilepsy regardless of etiology or syndrome.Multicenter retrospective study that evaluated the efficacy and safety of CBD for the treatment of drug resistant epilepsy of different etiologies in patients >2 years of age.Seventy-eight patients with a median age of 24 years and a wide spectrum of mainly structural and genetic etiologies were included. Patients were using a median of 3 antiseizure drugs (IQR=2-4) and had a median of 30 monthly seizures (IQR=12-100) before starting CBD. The median treatment time with CBD was 14 months (IQR=10-17). The efficacy analysis at the last available visit showed that mean percent reduction in seizures, ≥50% reduction in seizure frequency and seizure freedom was 67.8%, 68.8% and 11.5% respectively. We found no significant impact of concomitant clobazam use on the efficacy and safety of CBD. In the safety analysis, 28.2% (n = 22) of patients presented adverse events related to CBD and drug-retention rate was 78.2%.In a real-world setting, highly purified CBD has been shown to be safe and effective for the treatment of drug resistant epilepsy not related to Lennox-Gastaut syndrome, Dravet syndrome or Tuberous Sclerosis Complex. Based on these findings, highly purified CBD should be considered as an adjuvant therapy for drug resistant epilepsy, regardless of its underlying cause or specific syndrome. Nevertheless, this assumption should be validated through further controlled trials.

Religion and spirituality (R/S) serve as coping mechanisms for circumstances that threaten people's psychological well-being. However, using R/S inappropriately to deal with difficulties and problems in daily life may include the practice of Spiritual Bypass (SB). SB refers to avoiding addressing emotional problems and trauma, rather than healing and learning from them. On the other hand, coping strategies may be determined by the cultural context. This study aims to describe the presence of SB in individuals who may have experienced stressful situations and to understand the influence of culture on SB by comparing SB in two culturally different groups. The sample consists of a total of 435 people, 262 of Honduran nationality and 173 of Spanish nationality. Both groups are approximately equivalent in age and gender. The degree of SB, stressful events, perception of social support and spiritual well-being are examined, respectively, through the Spiritual Bypass Scale, and specific items and subscales from the Social Readjustment Rating Scale, Multidimensional Scale of Perceived Social Support, and the Functional Assessment of Chronic Illness Therapy - Spiritual Wellbeing. The results showed a higher spiritual well-being and use of SB in the Honduran sample as compared to the Spanish sample, but similar social support and stressful events. Furthermore, some of the factors predicting SB were different between the two samples. While age and a greater number of R/S practices were important in both samples, for the Honduran sample the variables that best explained SB were being a Christian, having greater social support, fewer stressful events, and greater attendance at church or temple. For the Spanish sample, however, the variable that best explained SB was studying R/S texts. Therefore, SB must be understood within the culture in which it develops, since in different cultural contexts it appears to relate to differing factors. Thus, SB becomes a possible functional or dysfunctional coping strategy depending on the social context.

The COVID-19 lockdown has had a massive psychological impact on mental health in the general population, with increases in anxiety, depression, and post-traumatic stress disorder. Spiritual well-being, specifically peace and meaning, has already been identified as one of the main protective factors for these disorders in the COVID-19 context. The aim of the present study is to identify facilitating elements for peace and meaning during the COVID-19 lockdown in Spain. Online surveys were used to obtain data from a sample of 3480 Spanish people. Self-compassion and social support were positively related with peace and meaning, while loneliness and perceived discrimination were negatively related. The model for peace and meaning was statistically significant, explaining 47% of the variance. The significant variables were self-kindness, family support, mindfulness, and sense of belonging having a positive association and loneliness a negative one.

Abstract Cellular reprogramming from somatic to pluripotent cells is the basis for multiple applications aimed to replace damaged tissues in regenerative medicine. However, this process is limited by intrinsic barriers that are induced in response to reprogramming factors. In this manuscript we report that miR-203, a microRNA with multiple functions in differentiation and tumor suppression, acts as an endogenous barrier to reprogramming. Genetic ablation of miR-203 results in enhanced reprogramming whereas its expression prevents the formation of pluripotent cells both in vitro and in vivo. Mechanistically, this effect correlates with the direct repression of NFATC2, a transcription factor involved in the early phases of reprogramming. Inhibition of NFATC2 mimics miR-203 effects whereas NFATC2 overexpression rescues inducible cell pluripotency in miR-203-overexpressing cultures. These data suggest that miR-203 repression may favor the efficiency of reprogramming in a variety of cellular models.

Abstract A hallmark of many malignant tumors is dedifferentiated (immature) cells bearing slight or no resemblance to the normal cells from which the cancer originated. Tumor dedifferentiated cells exhibit a higher capacity to survive to chemo and radiotherapies and have the ability to incite tumor relapse. Inducing cancer cell differentiation would abolish their self-renewal and invasive capacity and could be combined with the current standard of care, especially in poorly differentiated and aggressive tumors (with worst prognosis). However, differentiation therapy is still in its early stages and the intrinsic complexity of solid tumor heterogeneity demands innovative approaches in order to be efficiently translated into the clinic. We demonstrate here that microRNA 203, a potent driver of differentiation in pluripotent stem cells (ESCs and iPSCs), promotes the differentiation of mammary gland tumor cells. Combining mouse in vivo approaches and both mouse and human-derived tridimensional organoid cultures, we report here that miR-203 influences the self-renewal capacity, plasticity and differentiation potential of breast cancer cells, and prevents tumor cell growth in vivo. Our work sheds light on differentiation-based antitumor therapies and offers miR-203 as a promising tool for directly confronting the tumor-maintaining and regeneration capability of cancer cells.

Abstract A hallmark of many malignant tumors is dedifferentiated (immature) cells bearing slight or no resemblance to the normal cells from which the cancer originated. Tumor dedifferentiated cells exhibit a higher capacity to survive to chemo and radiotherapies and have the ability to incite tumor relapse. Inducing cancer cell differentiation would abolish their self-renewal and invasive capacity and could be combined with the current standard of care, especially in poorly differentiated and aggressive tumors (with worst prognosis). However, differentiation therapy is still in its early stages and the intrinsic complexity of solid tumor heterogeneity demands innovative approaches in order to be efficiently translated into the clinic. We demonstrate here that microRNA 203, a potent driver of differentiation in pluripotent stem cells (ESCs and iPSCs), promotes the differentiation of mammary gland tumor cells. Combining mouse in vivo approaches and both mouse and human-derived tridimensional organoid cultures, we report that miR-203 influences the self-renewal capacity, plasticity and differentiation potential of breast cancer cells and prevents tumor cell growth in vivo. Our work sheds light on differentiation-based antitumor therapies and offers miR-203 as a promising tool for directly confronting the tumor-maintaining and regeneration capability of cancer cells.

Additional file 2. Supplementary table, extended excel data for Supplementary Figure S4.

Abstract Background: ABTL0812 is a first-in-class orally administered compound currently in Phase I/Ib First in Human Clinical Trial in patients with advanced solid tumors (NCT02201823). ABTL0812 has cytotoxic effect on a wide range of human tumor cell lines, including those which have become resistant to standard therapy. We hereby dissect the anti-tumor activity of ABTL0812, which relies on a novel mechanism of action that promotes inhibition of the Akt/mTOR axis in cancer cells. Material &amp; methods: ABTL0812 molecular targets were identified by in silico analysis, comparing ABTL0812 chemical structure against a database including more than one million receptor-ligand interaction data. Functional relevance of the targets was confirmed biochemically and pharmacologically. ABTL0812 mechanism of action was established using human lung and pancreatic tumor cells, MEF KO cells, as well as tumor xenografts. Results: In silico screening showed that ABTL0812 binds four targets which regulate tumor progression through Akt/mTOR axis. Two of them are the transcription factors PPARα and PPARγ (Peroxisome-Proliferator Activating Receptors). In lung and pancreatic tumor cells ABTL0812 activated PPARα/γ-dependent gene transcription, while pharmacological inhibition with PPARα/γ antagonists impaired ABTL0812 cytotoxic effect. Interestingly, ABTL0812 induced transcription of the endogenous Akt inhibitor TRIB3 (tribbles homologue 3) through PPARα/γ activation. TRIB3 is a pseudokinase that inhibits Akt by direct binding and preventing its phosphorylation by mTORC2 complex. According to this, ABTL0812-induced TRIB3 overexpression resulted in inhibition of Akt phosphorylation, impaired phosphorylation of the Akt substrates TSC2 and PRAS40 and mTORC1 inhibition (pS6), which in turn promoted autophagy-mediated tumor cell death. MEF TRIB3-/- cells were resistant to ABTL0812-induced cell death, indicating that TRIB3 mediates ABTL0812 citotoxicity. Finally, Akt inhibition was observed in human lung and pancreatic tumor xenograft models treated with ABTL0812 and in human platelets incubated with ABTL0812. This supported the rational for using Akt phosphorylation as a pharmacodynamic biomarker to monitor activity of ABTL0812 in patients included in the Clinical Trial. Conclusion: ABTL0812 promotes autophagy-mediated cell death in different cancer paradigms by inhibiting the Akt/mTOR axis through a novel mechanism of action. ABTL0812 induces PPARα/γ-mediated transcription of the TRIB3 gene. Upregulated TRIB3 protein binds and inhibits Akt, leading to mTORC1 inhibition and reduction of tumor growth. These findings provide evidences that ABTL0812 may be an effective therapeutic strategy for targeting cancer. Citation Format: Tatiana Erazo, Mariana Gomez-Ferreria, Jose Alfon, Mar Lorente, Maria Salazar, Anna Lopez, Marc Cortal, Pau Munoz-Guardiola, Pedro Gascon, Guillermo Velasco, Carles Domenech, Jose M. Lizcano. ABTL0812, a new antitumor drug that inhibits the axis Akt/mTOR through a novel mechanism of action. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 672. doi:10.1158/1538-7445.AM2015-672

Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. When these tumors are in advanced stages, few therapeutic options are available. Therefore, it is essential to search for new treatments to fight this disease. In this study, we investigated the effects of cannabinoids – a novel family of potential anticancer agents – on the growth of HCC. We found that D 9 -tetrahydrocannabinol (D 9 -THC, the main active component of Cannabis sativa) and JWH-015 (a cannabinoid receptor 2 (CB2) cannabinoid receptor-selective agonist) reduced the viability of the human HCC cell lines HepG2 (human hepatocellular liver carcinoma cell line) and HuH-7 (hepatocellular carcinoma cells), an effect that relied on the stimulation of CB2 receptor. We also found that D 9 -THC- and JWH-015-induced autophagy relies on tribbles homolog 3 (TRB3) upregulation, and subsequent inhibition of the serine–threonine kinase Akt/mammalian target of rapamycin C1 axis and adenosine monophosphate-activated kinase (AMPK) stimulation. Pharmacological and genetic inhibition of AMPK upstream kinases supported that calmodulin-activated kinase kinase b was responsible for cannabinoid-induced AMPK activation and autophagy. In vivo studies revealed that D 9 -THC and JWH-015 reduced the growth of HCC subcutaneous xenografts, an effect that was not evident when autophagy was genetically of pharmacologically inhibited in those tumors. Moreover, cannabinoids were also able to inhibit tumor growth and ascites in an orthotopic model of HCC xenograft. Our findings may contribute to the design of new therapeutic strategies for the management of HCC. Cell Death and Differentiation (2011) 18, 1099–1111; doi:10.1038/cdd.2011.32; published online 8 April 2011 Hepatocellular carcinoma (HCC) is one of the most common solid tumors and the third leading cause of cancer-related death worldwide. 1 Its prognosis remains reserved, with a 5-year survival rate of o5%. 2 It is the most common cause of death in patients with cirrhosis 3 and, according to the World Health Organization, the incidence of HCC is expected to increase until 2030. The overall survival of patients with HCC has not significantly improved in the past two decades. Current treatments are only applicable at early stages of tumor development and include tumor resection, liver transplantation, chemoembolization and sorafenib administration. 4 However, approximately half of the patients suffer tumor recurrence. The most important mechanism of liver cancer progression is cell proliferation. Although in recent years several clinical trials have tested the efficacy of agents that selectively target important signaling pathways involved in the control of this process, no relevant improvement in the prognostic/survival of patients with HCC has been achieved

Cannabinoid receptor (CBs) agonists affect the growth of tumor cells via activation of deadly cascades. The spectrum of action of these agents and the precise role of the endocannabinoid system (ECS) on oncogenic processes remain elusive. Herein we compared the effects of synthetic (CP 55-940 and WIN 55,212-2) and endogenous (anandamide or AEA) CBs agonists (10–20 μM) on morphological changes, cell viability, and induction of apoptosis in primary astrocytes and in two glioblastoma cell lines (C6 and U373 cells) in order to characterize their possible differential actions on brain tumor cells. None of the CBs agonist tested induced changes in cell viability or morphology in primary astrocytes. In contrast, CP 55-940 significantly decreased cell viability in C6 and U373 cells at 5 days of treatment, whereas AEA and WIN 55,212-2 moderately decreased cell viability in both cell lines. Treatment of U373 and C6 for 3 and 5 days with AEA or WIN 55,212-2 produced discrete morphological changes in cell bodies, whereas the exposure to CP 55-940 induced soma degradation. CP 55-940 also induced apoptosis in both C6 and U373 cell lines. Our results support a more effective action of CP 55-940 to produce cell death of both cell lines through apoptotic mechanisms. Comparative aspects between cannabinoids with different profiles are necessary for the design of potential treatments against glial tumors.

Stemming from one creative experience that emerged in London during the lockdown period of early 2020, called the “Emergency Festival”, this article is a result of observations based on practice, centred around the festival that a group of multicultural, interdisciplinary movement-based researchers and dancers created, curated, and participated in. It explores the possibility of making a radical alterity out of a hitherto previously established ideas of territory, time, and community, using performative writing as practice-based analysis scheme. Employing the concept of “communitas” by Victor Turner (1969) to approach the phenomenon of dance through distance, the article examines the importance of the emergence of collaboration as a way forward, epistemologically looking at dance as a method of creating and sustaining communities that are longing for a sense of home in times of change. The writing is divided into three parts, focussing on the aspects of space, time, and community, all the while embedded in the nature of movement and its effect on the practitioners, and onlookers, concluding with contemplation on the place of dance in varied mediums and the way forward to study it in a period of global disruption.