Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells

© The Author(s) 2020Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7–9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.We acknowledge and thank Dr. Nuria Malats and Jaime Villarreal from the Spanish National Cancer Research Center (CNIO) for RNA sequencing and analysis, funded by Fondo de Investigaciones Sanitarias (FIS) grant PI18/01347. We thank Patricia Sánchez-Tomero and Marina Ochando-Garmendia for technical assistance and support and Dr. Raúl Sánchez Lanzas for assistance with autophagy experiments. We want to particularly acknowledge the patients and the BioBank Hospital Ramón y Cajal-IRYCIS (PT13/0010/0002) integrated in the Spanish National Biobanks Network for its collaboration and, in particular, Adrián Povo Retana for macrophage isolation. We would also like to thank the Transmission Electron Microscopy Unit Laboratory, part of the UAM Interdepartmental Investigation Service (SIdI); Coral Pedrero for exceptional help with in vivo experiments; and the laboratories of Dr. Amparo Cano and Dr. José González Castaño for reagents and helpful discussions. S.V. was a recipient of an Ayuda de Movilidad del Personal Investigador del IRYCIS, a mobility grant from the Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain, and a pre-doctoral fellowship from the Comunidad de Madrid, Ayudas Para La Contratación De Investigadores Predoctorales Y Posdoctorales (PEJD-2017-PRE/BMD-5062), Madrid, Spain. This study was supported by a Rámon y Cajal Merit Award (RYC-2012-12104) from the Ministerio de Economía y Competitividad, Spain (to B.S.); funding from la Beca Carmen Delgado/Miguel Pérez-Mateo from AESPANC-ACANPAN Spain (to B.S.); a Conquer Cancer Now Grant from the Concern Foundation (Los Angeles, CA, USA) (to B.S.); a Coordinated grant (GC16173694BARB) from the Fundación Asociación Española Contra el Cáncer (AECC) (to B.S.); FIS grants PI18/00757 (to B.S.), PI16/00789 (to M.A.F.-M.), PI18/00267 (to L.G.-B.; co-financed through Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa”); a Miguel Servet award (CP16/00121) (to P.S.); a Max Eder Fellowship of the German Cancer Aid (111746) (to P.C.H.); and the German Research Foundation (DFG, CRC 1279 “Exploiting the human peptidome for Novel Antimicrobial and Anticancer Agents”; to P.C.H.)

Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma

[Objective]: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models.[Design]: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation.[Results]: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment.[Conclusion]: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.JCL-G received support from a 'la Caixa' Foundation (ID 100010434) fellowship (LCF/BQ/DR21/11880011). This study was supported by ISCIII FIS grants PI18/00757 and PI21/01110 (BSJ) and PI18/00267 (LG-B), and grants from the Spanish Ministry of Economy and Innovation SAF2016-76504-R (ACan and FP), PID2019-111052RB-I00 (FP), PID2019-104644RB-I00 (GM-B), a Ramón y Cajal Merit Award RYC-2012–12104 (BSJ) and ISCIII, CIBERONC, CB16/12/00446 (ACar) and CB16/12/00295 (ACan and GM-B), all of them co-financed through Fondo Europeo de Desarrollo Regional (FEDER) 'Una manera de hacer Europa'; a Fero Foundation Grant (BSJ); a Coordinated grant (GC16173694BARB) from the Fundación Científica Asociación Española Contra el Cáncer (FC-AECC) (BSJ); a Miguel Servet award (CP16/00121) (PS); a DFG, German Research Foundation Grant—Project no: 492 436 553 (KG); and a Max Eder Fellowship of the German Cancer Aid (111746) (PCH

The Effects of PMM2-CDG-Causing Mutations on the Folding, Activity, and Stability of the PMM2 Protein

© 2015 WILEY PERIODICALS, INC.. Congenital disorder of glycosylation type Ia (PMM2-CDG), the most common form of CDG, is caused by mutations in the PMM2 gene that reduce phosphomannomutase 2 (PMM2) activity. No curative treatment is available. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The effects of the mutations on PMM2/Pmm2 stability, oligomerization, and enzyme activity were explored in an optimized bacterial system. The mutant proteins were associated with an enzymatic activity of up to 47.3% as compared with wild type (WT). Stability analysis performed using differential scanning fluorimetry and a bacterial transcription-translation-coupled system allowed the identification of several destabilizing mutations (p.V44A, p.D65Y, p.R123Q, p.R141H, p.R162W, p.F207S, p.T237M, p.C241S). Exclusion chromatography identified one mutation, p.P113L, that affected dimer interaction. Expression analysis of the p.V44A, p.D65Y, p.R162W, and p.T237M mutations in a eukaryotic expression system under permissive folding conditions showed the possibility of recovering their associated PMM2 activity. Together, the results suggest that some loss-of-function mutations detected in PMM2-CDG patients could be destabilizing, and therefore PMM2 activity could be, in certain cases, rescuable via the use of synergetic proteostasis modulators and/or chaperones. The present work describes the functional analysis of nine human PMM2 mutant proteins frequently found in PMM2-CDG patients and also two murine Pmm2 mutations carried by the unique PMM2-CDG mouse model described to overcome embryonic lethality. The results suggest that PMM2-CDG could be considered a conformational disease and therefore PMM2 activity could be rescuable via the use of small stabilizer molecules. Unfortunately, this mouse model is inadequate for testing that kind of compounds since neither mutation causes conformational instability.Peer Reviewe

Evaluation and Design of Video Games: Creating Objects of Learning in Communities of Practice

Los videojuegos superan los contextos de ocio y se pueden considerar como hipertextos lúdicos, que configuran modelos de aprendizaje y favorecen el desarrollo y adquisición de determinadas competencias y habilidades mediante la formulación de prácticas formativas que transcienden a las tradiciones. En el presente artículo se pone de manifiesto la aplicabiblidad de los videojuegos desde el punto de vista educativo, ofreciendo a los docentes orientaciones para su incorporación en el aula. Se describe, de igual modo, una herramienta de ayuda para la evaluación de videojuegos con el fin de facilitar su integración curricular y aprovechamiento didáctico a través de tres grandes dimensiones de análisis que pueden ser utilizadas, a su vez, para el diseño de videojuegos educativos. En el documento, se presentan diferentes herramientas educativas para el diseño de videojuegos, que pueden ser exportados como objetos de aprendizaje facilitando su reutilización y transferencia a otros contextos y situaciones formativas. Compartir e intercambiar estos objetos de aprendizaje de carácter lúdico a través de comunidades de práctica supone para el profesorado un gran apoyo y una fuente de recursos importante.The videogames and leisure contexts can be considered as ludic hypertexts, learning models that shape and enhance the development and acquisition of certain competences and abilities through the development of pedagogical practices that transcend school proposals traditions The article presents the application of videogames from the field of education, providing valuable guidance to teachers for inclusion in the classroom. Described, similarly, a tool for evaluation of video games is described, in order to facilitate integration and use didactic curriculum by three dimensions of study. The dimensions can be used also for educational game design. In the document, there are different educational tools for game design, which can be exported as learning objects facilitating their reuse and transfer to other contexts and training situations. Sharing and exchanging learning objects through communities of practice for teachers are very supportive and provide a source of important resources

Evaluación y diseño de videojuegos: generando objetos de aprendizaje en comunidades de práctica

The videogames and leisure contexts can be considered as ludic hypertexts, learning models that shape and enhance the development and acquisition of certain competences and abilities through the development of pedagogical practices that transcend school proposals traditions The article presents the application of videogames from the field of education, providing valuable guidance to teachers for inclusion in the classroom. Described, similarly, a tool for evaluation of video games is described, in order to facilitate integration and use didactic curriculum by three dimensions of study. The dimensions can be used also for educational game design. In the document, there are different educational tools for game design, which can be exported as learning objects facilitating their reuse and transfer to other contexts and training situations. Sharing and exchanging learning objects through communities of practice for teachers are very supportive and provide a source of important resources.Los videojuegos superan los contextos de ocio y se pueden considerar como hipertextos lúdicos, que configuran modelos de aprendizaje y favorecen el desarrollo y adquisición de determinadas competencias y habilidades mediante la formulación de prácticas formativas que transcienden a las tradiciones. En el presente artículo se pone de manifiesto la aplicabiblidad de los videojuegos desde el punto de vista educativo, ofreciendo a los docentes orientaciones para su incorporación en el aula. Se describe, de igual modo, una herramienta de ayuda para la evaluación de videojuegos con el fin de facilitar su integración curricular y aprovechamiento didáctico a través de tres grandes dimensiones de análisis que pueden ser utilizadas, a su vez, para el diseño de videojuegos educativos. En el documento, se presentan diferentes herramientas educativas para el diseño de videojuegos, que pueden ser exportados como objetos de aprendizaje facilitando su reutilización y transferencia a otros contextos y situaciones formativas. Compartir e intercambiar estos objetos de aprendizaje de carácter lúdico a través de comunidades de práctica supone para el profesorado un gran apoyo y una fuente de recursos importante

Lysyl oxidase-like 3 in melanoma progression

Resumen del trabajo presentado al 1st PhD Research Symposium in Health Sciences and Biomedicine, celebrado en la Universidad Autónoma de Madrid el 18 de mayo de 2018.SAF2013-44739R, SAF2016-76504-R Ministerio de Economía y Competitividad (MINECO), CB16/12/00295 CIBERONC, 16-0295 Worldwide Cancer Research (WWCR), Red de Cáncer RETIC-RD12/0036/0007 (Instituto de Salud Carlos III).Peer reviewe

Análisis de posibles proteínas que interaccionan con la fukutina

Póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.La fukutina, una proteína codificada por el gen FKTN, se ha relacionado con un conjunto de enfermedades congénitas de carácter recesivo denominadas actualmente como distrofias musculares-distroglicanopatías (MDDG, de sus siglas en inglés). La falta de función de la fukutina conduce a la hipoglicosilación del alfa-distroglicano (α-DG). Esta proteína actúa como anclaje de la célula a la matriz extracelular (MEC) en diferentes tejidos y órganos, sobre todo músculo, cerebro y retina. El α-DG sufre importantes y masivas O-glicosilaciones postraduccionales en residuos de serinas/treoninas de su conocido “dominio mucina”, generando diferentes tipos de cadenas tanto de O-manosilglicanos como de O-N-acetilgalactosaminilglicanos. A través de estos residuos se realiza la interacción con proteínas de la MEC como laminina, agrina y perlecano, así como con las proteínas sinápticas neurexina y pikachurina. Sin embargo, hasta la fecha, la función de la fukutina en la O-glicosilación del α-DG es desconocida. En un intento de elucidar su función, en este trabajo hemos procedido a sobreexpresar esta proteína en la línea celular humana HEK293T, purificarla y analizar mediante espectrometría de masas las proteínas que coeluyen con ella. Hemos seleccionado una serie de proteínas potencialmente interesantes, centrándonos preferentemente el estudio de la proteína CMAS (citidina monofosfo-N-ácido acetilneuramínico sintetasa). Esta enzima cataliza la síntesis de CMP-Neu5Ac, un sustrato utilizado por diferentes si aliltransferasas para la adición de ácido siálico, el cual está presente en muchas de las cadenas de O-glicanos del α-DG.Instituto de Salud Carlos III, proyecto PI12/0157.Peer reviewe

QEAVis: Evaluación Cuantitativa de la Visibilidad de los Sitios Web Académicos

El proyecto plantea la aplicación de las TLH a un problema importante como es medir la visibilidad académica en la web, sentando las bases de una evaluación cuantitativa del compromiso de los departamentos universitarios con la accesibilidad pública de su información. Para ello es necesario desarrollar indicadores web (Cibermetría) y estudiar la visibilidad de los sitios web académicos, haciendo especial énfasis en la presencia del español (de importancia estratégica) y en el ámbito de las disciplinas relacionadas con humanidades (que requiere una ayuda especial respecto a su posicionamiento en web).The project proposes the application of HLT to an important problem such as the measurement of the academic visibility in the web, giving the basis of a quantitative evaluation of the universities departments’ commitment in the public access to their information. Web indicators (Cybermetrics) must be developed and applied to the study of the academic websites visibility, with special focus on the presence of the Spanish language (of strategic importance) and the academic areas related to humanities (which need special help for their web positioning).Financiado por el Ministerio de Ciencia e Innovación TIN2007-67581

Protein misfolding diseases: Prospects of pharmacological treatment

Protein misfolding has been linked to numerous inherited diseases. Loss- and gain-of-function mutations (common features of genetic diseases) may cause the destabilization of proteins, leading to alterations in their properties and/or cellular location, resulting in their incorrect functioning. Misfolded proteins can, however, be rescued via the use of proteostasis regulators and/or pharmacological chaperones, suggesting that treatments with small molecules might be developed for a range of genetic diseases. This work describes the potential of these small molecules in this respect, including for the treatment of congenital disorder of glycosylation (CDG) due to phosphomannomutase 2 deficiency (PMM2-CDG).This work was funded by the Fondo Investigación Sanitaria, grants PI13/01239 and PI16/00573; the Fundación Isabel Gemio, an institutional grant from the Fundación Ramón Areces and by the European Regional Development Fund. The authors thank all the PMM2-CDG families involved in the research undertaken at our laboratoryPeer reviewe

RED. Revista de educación a distancia

Monográfico con el título: 'Sobre vídeojuegos y aprendizaje'. Resumen basado en el de la publicaciónSe aborda el tema de los videojuegos desde el punto de vista educativo para su incorporación en el aula. Se describe una herramienta para la evaluación de videojuegos con el fin de facilitar su integración curricular y aprovechamiento didáctico a través de tres grandes dimensiones de análisis que pueden ser utilizadas, a su vez, para el diseño de videojuegos educativos. Se presentan diferentes herramientas educativas para el diseño de videojuegos, que pueden ser exportados como Objetos de Aprendizaje (OA) facilitando su reutilización y transferencia a otros contextos y situaciones formativas. Se aportan ejemplos de plataformas que comparten e intercambian estos Objetos de Aprendizaje de carácter lúdico a través de comunidades de práctica, lo que supone un gran apoyo y una fuente de recursos importante para el profesorado.MurciaBiblioteca de Educación del Ministerio de Educación, Cultura y Deporte; Calle San Agustín, 5 - 3 planta; 28014 Madrid; Tel. +34917748000; [email protected]