Modelos celulares de cáncer de próstata

Modelos celulares de cáncer de próstata. Líneas celulares, PC-3/S-luc y PC-3/M-luc, derivadas de la línea de cáncer de próstata PC-3, modificadas de forma que expresan el gen luciferasa (luc) para el desarrollo de un modelo de cáncer, útil para el diagnóstico y/o pronóstico de cáncer, específicamente de próstata, determinando la diferente expresión génica de los marcadores biológicos de invasividad y metástasis. Así como, un método in vitro de búsqueda de agentes para tratamiento o prevención del cáncer.Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Regulation of self-renewal in colorectal cancer cell models

[spa] El factor de transcripción SOX2 es una proteína clave implicada en las propiedades adscritas a células madre y también está implicado en la iniciación, mantenimiento y progresión tumoral en distintos tipos de cáncer. Mediante estudio in vitro de las consecuencias fenotípicas de la sobreexpresión y represión de SOX2 en un grupo de líneas celulares, nos hemos centrado en el estudio de dos lineas celulares isogénicas de cáncer colorectal (SW620 y SW480) que presentan una marcada expresión diferencial de SOX2 que correlaciona positivamente con su agresividad. En conjunto, hemos encontrado que altos niveles de SOX2 induce daño al DNA e inducción de especies reactivas de oxígeno, arresto en ciclo celular y apoptosis. También hemos determinado que la represión de SOX2 en la línea metastásica de cáncer colorrectal SW620, induce arresto en G1 y que éste depende parcialmente en los inhibidores de ciclina dependiente de kinasa p21 y p27, junto a una drástica reducción en la formación de esferoides en condiciones independientes de anclaje a sustrato y de tumorigenicidad in vivo. Además, los análisis con plásmidos reporteros combinando represión y sobreexpresión de SOX2 sugieren que este factor de transcripción podría actuar como un regulador transcripcional negativo de p27. A su vez, la represión de p27 es capaz de revertir parcialmente el arresto en ciclo celular en G1 inducido por la represión de SOX2 pero no restablece el crecimiento de esferoides en condiciones independientes de anclaje a sustrato. De manera destacable, el silenciamiento de SOX2 induce la bajada de expresión de los niveles de LGR5, un conocido marcador de células madre adultas intestinales, aunque no se ha podido determinar en este estudio si esta regulación está mediada directamente por SOX2 o por otros mecanismos. De igual modo, la represión de LGR5 en la línea SW620 también produce una reducción significativa en la capacidad de formar esferoides in vitro y de crecer tumores in vivo, a pesar de incrementar de manera relevante la actividad transcripcional de la β-catenina. El incremento de la actividad transcripcional de la β-catenina al reprimir LGR5, es concomitante a la reducción de la fracción de esta proteína unida a membrana y al aumento de su translocación nuclear pero no a la acumulación de ésta o a la bajada de expresión de los reguladores negativos de la ruta canónica de Wnt, ZNRF3 o RNF43. Estos resultados indican que LGR5 es un regulador negativo de la ruta canónica de la señalización Wnt en ausencia de sus ligandos conocidos (R-spondinas), y que la elevada actividad transcripcional del complejo formado por β-catenina/TCF/LEF no implica necesariamente el incremento del potencial de auto-renovación en los modelos celulares de cáncer colorrectal estudiados.[eng] The transcription factor SOX2 is a key determinant of stem cell properties, also involved in the initiation, maintenance and progression of several human cancers. By studying the in vitro phenotypic consequences of SOX2 overexpression and knock down in a panel of cell lines, we have found that high levels of SOX2 leads to DNA damage and accumulation of reactive oxygen species, accompanied with cell cycle arrest and apoptosis in colorectal cancer cell lines, SW620 and SW480. We have also found that knock down of SOX2 in the metastatic colorectal cancer cell line SW620, led to G1 cell cycle arrest partially dependent on the cyclin-dependent kinase inhibitors p21 and p27, as well as abrogation of anchorage-independent growth in vitro and tumor growth in vivo. Knock down of SOX2 caused the upregulation of p27 and reporter assays combining knock down and overexpression of this transcription factor suggest that SOX2 may act as a negative transcriptional regulator of p27. Knock down of p27 partially reverted the cell-cycle arrest induced by SOX2 repression but was insufficient to restore anchorage-independent growth in vitro. In addition, it also led to downregulation of LGR5 transcript levels, a known intestinal stem cell marker, but it is unknown whether this regulation is mediated directly by SOX2 or by other mechanisms. Knock down of LGR5 partially phenocopied the repression of SOX2 in SW620 cells; it abrogated their anchorage-independent growth in vitro and tumor growth in vivo, in spite of a strong induction of β-catenin transcriptional activity. Activation of β-catenin transcriptional activity upon LGR5 knock down was paralleled by decreased membrane association and enhanced nuclear translocation but not by β-catenin protein accumulation or downregulation of the Wnt/β-catenin negative regulators ZNRF3 or RNF43. These results indicate that LGR5 is a negative regulator of canonical Wnt signaling in the absence of their cognate ligands (R-spondins) and that high β-catenin/TCF/LEF transcriptional activity does not involve necessarily increase of self-renewal potential in these colorectal cancer cell models studied

A key role for transketolase-like 1 in tumor metabolic reprogramming

Metabolic reprogramming, a crucial cancer hallmark, shifts metabolic pathways such as glycolysis, tricarboxylic acid cycle or lipogenesis, to enable the growth characteristics of cancer cells. Here, we provide evidence that transketolase-like 1 (TKTL1) orchestrates aerobic glycolysis, fatty acid and nucleic acid synthesis, glutamine metabolism, protection against oxidative stress and cell proliferation. Furthermore, silencing of TKTL1 reduced the levels of sphingolipids such as lactosylceramide (a sphingolipid regulating cell survival, proliferation and angiogenesis) and phosphatidylinositol (which activates PI3K/Akt/mTOR signaling). Thus, in addition to its well-known roles in glucose and amino acid metabolism, TKTL1 also regulates lipid metabolism. In conclusion, our study provides unprecedented evidence that TKTL1 plays central roles in major metabolic processes subject to reprogramming in cancer cells and thus identifies TKTL1 as a promising target for new anti-cancer therapies.This work was supported by grants to M.C. (Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) – Generalitat de Catalunya, 2014SGR1017; ICREA Academia from the ICREA foundation; and MINECO-European Commission FEDER – Una manera de hacer Europa, SAF2014-56059-R), T.M.T. (MINECO-European Commission FEDER – Una manera de hacer Europa, SAF2015-66984-C2-1-R; and Xarxa de Referència en Biotecnologia) and P.C. (Research Foundation-Flanders (FWO), Belgian Science Policy grant (IUAP P7/03); long-term structural Methusalem funding by the Flemish Government; European Research Council (ERC) Advanced Research Grant (EU-ERC269073); and AXA Research Fund).Peer Reviewe

SPARC mediates metastatic cooperation between CSC and non-CSC prostate cancer cell subpopulations

Francesca Mateo et al.[Background] Tumor cell subpopulations can either compete with each other for nutrients and physical space within the tumor niche, or co-operate for enhanced survival, or replicative or metastatic capacities. Recently, we have described co-operative interactions between two clonal subpopulations derived from the PC-3 prostate cancer cell line, in which the invasiveness of a cancer stem cell (CSC)-enriched subpopulation (PC-3M, or M) is enhanced by a non-CSC subpopulation (PC-3S, or S), resulting in their accelerated metastatic dissemination. Methods: M and S secretomes were compared by SILAC (Stable Isotope Labeling by Aminoacids in Cell Culture). Invasive potential in vitro of M cells was analyzed by Transwell-Matrigel assays. M cells were co-injected with S cells in the dorsal prostate of immunodeficient mice and monitored by bioluminescence for tumor growth and metastatic dissemination. SPARC levels were determined by immunohistochemistry and real-time RT-PCR in tumors and by ELISA in plasma from patients with metastatic or non-metastatic prostate cancer.[Results] Comparative secretome analysis yielded 213 proteins differentially secreted between M and S cells. Of these, the protein most abundantly secreted in S relative to M cells was SPARC. Immunodepletion of SPARC inhibited the enhanced invasiveness of M induced by S conditioned medium. Knock down of SPARC in S cells abrogated the capacity of its conditioned medium to enhance the in vitro invasiveness of M cells and compromised their potential to boost the metastatic behavior of M cells in vivo. In most primary human prostate cancer samples, SPARC was expressed in the epithelial tumoral compartment of metastatic cases.[Conclusions] The matricellular protein SPARC, secreted by a prostate cancer clonal tumor cell subpopulation displaying non-CSC properties, is a critical mediator of paracrine effects exerted on a distinct tumor cell subpopulation enriched in CSC. This paracrine interaction results in an enhanced metastatic behavior of the CSC-enriched tumor subpopulation. SPARC is expressed in the neoplastic cells of primary prostate cancer samples from metastatic cases, and could thus constitute a tumor progression biomarker and a therapeutic target in advanced prostate cancer. © 2014 Mateo et al.; licensee BioMed Central Ltd.FM is a Juan de la Cierva Investigator, OM is a FPU Fellow from the Spanish Ministries of Science and Innovation (MICINN) and Economy and Competitiveness (MINECO) and LS is the recipient of a IDIBAPS fellowship. This work was supported by grants to TMT from MICINN (SAF2011-24686) and MINECO (SAF2012-40017-C02-01), Catalan Agència d’Ajuts Universitaris i de Recerca (AGAUR; 2009SGR1482), and Xarxa de Referència en Biotecnologia; to RP from Instituto de Salud Carlos III (ISCIII; RETICS RD06-0020/0058) and MICINN (SAF2011-30496); to IA from MINECO (PI11/079 and IPT-090000-2010-001; the latter cofinanced by the European Regional Development Fund) and AGAUR (SGR157); and to PLF from MICINN (FIS-PI080274) and MINECO (SAF2012-40017-C02-02). The Hospital Clínic-IDIBAPS Biobank is part of the Xarxa de Bancs de Tumors de Catalunya (XBTC), financed by the Pla Director d’Oncologia de Catalunya, and the Red Nacional de Biobancos (RNBB, ReTBioH), financed by the ISCIII (RETICS RD09-0076/0038)We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer Reviewe

CRISPR/Cas9-Mediated Knockin Application in Cell Therapy: A Non-viral Procedure for Bystander Treatment of Glioma in Mice

The use of non-viral procedures, together with CRISPR/Cas9 genome-editing technology, allows the insertion of single-copy therapeutic genes at pre-determined genomic sites, overcoming safety limitations resulting from random gene insertions of viral vectors with potential for genome damage. In this study, we demonstrate that combination of non-viral gene delivery and CRISPR/Cas9-mediated knockin via homology-directed repair can replace the use of viral vectors for the generation of genetically modified therapeutic cells. We custom-modified human adipose mesenchymal stem cells (hAMSCs), using electroporation as a transfection method and CRISPR/Cas9-mediated knockin for the introduction and stable expression of a 3 kb DNA fragment including the eGFP (selectable marker) and a variant of the herpes simplex virus 1 thymidine kinase genes (therapeutic gene), under the control of the human elongation factor 1 alpha promoter in exon 5 of the endogenous thymidine kinase 2 gene. Using a U87 glioma model in SCID mice, we show that the therapeutic capacity of the new CRISPR/Cas9-engineered hAMSCs is equivalent to that of therapeutic hAMSCs generated by introduction of the same therapeutic gene by transduction with a lentiviral vector previously published by our group. This strategy should be of general use to other applications requiring genetic modification of therapeutic cells. © 2017 The Author(s)This work was supported by MINECO/FEDER (grants SAF2015-64927-C2-1-R and SAF2015-64927-C2-2-R ) and the Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Terapia Celular-TERCEL) . We would like to thank M.A. Pastor and C. Bestard (flow cytometry) for their excellent technical assistance.Peer reviewe

Targeting of replicating CD133 and OCT4/SOX2 expressing glioma stem cells selects a cell population that reinitiates tumors upon release of therapeutic pressure

The existence of radio- and chemotherapy-surviving cancer stem cells is currently believed to explain the inefficacy of anti-glioblastoma (GBM) therapies. The aim of this study was to determine if a therapeutic strategy specifically targeting GBM stem cells (GSC) would completely eradicate a GBM tumor. In both the in vitro and the in vivo models, ganciclovir therapy targeting proliferating GSC promotes the survival of a quiescent, stem-like cell pool capable of reproducing the tumor upon release of the therapeutic pressure. Images of small niches of therapy-surviving tumor cells show organized networks of vascular-like structures formed by tumor cells expressing CD133 or OCT4/SOX2. These results prompted the investigation of tumor cells differentiated to endothelial and pericytic lineages as a potential reservoir of tumor-initiating capacity. Isolated tumor cells with pericyte and endothelial cell lineage characteristics, grown under tumorsphere forming conditions and were able to reproduce tumors after implantation in mice. © 2019, The Author(s).This work was supported by MINECO/FEDER (SAF2015-64927-C2-1-R) and the Instituto de Salud Carlos III (Red Temática de Investigación Cooperativa en Terapia Celular-TERCEL) and CIBER Cardiovascular -(CB16/11/00403) as part of the Plan Nacional de I + D + I cofounded by ISCIII-Sudirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER).Peer reviewe

Nanopartículas multicomponente y uso de las mismas

La invención se refiere a nanopartículas multicomponente para la transfección y selección de células . Las nanopartículas comprenden (a) nanopartículas de óxido de hierro superparamagnético (SPION), (b) un polímero que es un poli (beta-amino éster) (pBAE) modificado en los extremos con al menos un oliqopéptido y que tiene una masa molecular de entre 1.000 y 10.000 Da y (e) un polinucleótido. Estas partículas multicomponente potencian la eficiencia de transfección de la administración qénica no viral en diferentes tipos de células. Además, la característica magnética de estas innovadoras nanopartículas permite la rápida y masiva separación de células que contienen partículas mediante la aplicación de un campo magnético fuertePeer reviewedConsorcio Centro de Investigación Biomédica en Red, M.P., Sagetis Biotech SL, Consejo Superior de Investigaciones Científicas (España), Institut Quimic De Sarria CETS Fundacio PrivadaA1 Solicitud de patente con informe sobre el estado de la técnic

Metabolic Reprogramming and Dependencies Associated with Epithelial Cancer Stem Cells Independent of the Epithelial-Mesenchymal Transition Program

Esther Aguilar et al.In solid tumors, cancer stem cells (CSCs) can arise independently of epithelial-mesenchymal transition (EMT). In spite of recent efforts, the metabolic reprogramming associated with CSC phenotypes uncoupled from EMT is poorly understood. Here, by using metabolomic and fluxomic approaches, we identify major metabolic profiles that differentiate metastatic prostate epithelial CSCs (e-CSCs) from non-CSCs expressing a stable EMT. We have found that the e-CSC program in our cellular model is characterized by a high plasticity in energy substrate metabolism, including an enhanced Warburg effect, a greater carbon and energy source flexibility driven by fatty acids and amino acid metabolism and an essential reliance on the proton buffering capacity conferred by glutamine metabolism. An analysis of transcriptomic data yielded a metabolic gene signature for our e-CSCs consistent with the metabolomics and fluxomics analyses that correlated with tumor progression and metastasis in prostate cancer and in 11 additional cancer types. Interestingly, an integrated metabolomics, fluxomics, and transcriptomics analysis allowed us to identify key metabolic players regulated at the post-transcriptional level, suggesting potential biomarkers and therapeutic targets to effectively forestall metastasis.This work was supported by funds to M.C. from MICINN (SAF2011–25726 and SAF2014-56059-R, European Comission FEDER-Una manera de hacer Europa); Agència Catalana d'Ajuts Universitaris i de Recerca (AGAUR) (2014SGR-1017), ICREA Foundation (Generalitat de Catalunya) and European Commission (Metaflux, PITN-GA-2010-264780); to T.M.T. from MICINN (SAF2011-24686), MINECO (SAF2012-40017-C02-01, European Comission FEDER-Una manera de hacer Europa), AGAUR (2009SGR1482), and Xarxa de Referència en Biotecnologia; and to D.H. and F.M. from NIH (5R01CA158921-02). E.A. was supported by a fellowship from the MECD and a travel grant from RTICC; I.M.M. by a EC Marie Curie grant (Metaflux, PITN-GA-2010-264780)Peer Reviewe

Amidas de 2-amino-1,3-propanodioles y su uso como inhibidores de ceramidasas

[EN] The invention relates to a compound having formula (I) or a stereoisomer, a salt or a solvate, in which: A is -CH(OH)- or -C(=0)-; Z is H or OH; n is 0 or 1; R1 is alkyl(C1-C30), alkenyl(C2-C30) or alkynyl(C2-C30); B is -H, -N3 or -C?CH; R2 is -NHR3 or maleimide; R3 is -COR4, -COCOR4 or -SO2R4; and R4 is alkyl(C1-C16), alkenyl(C2-C16), alkynyl(C2-C16), epoxide or aziridine, on the condition that: a) when A is -CH(OH) and B is H, R3 is different from -COR4, R4 being alkyl(C1-C16); b) when A is -CH(OH), B is H and R3 is -COCOR4,R4 beingalkyl(C6), R1 is different from -CH=CH2-alkyl(C12), -C?CH-alkyl(C12) or alkyl(C13-C15); or c) when A is -C(=O), R1 is alkenyl(C2-C30), B is H and n is 0, R3 is different from -COR4, R4 being alkyl(C1C16). The invention also relates to the use of the compound having formula (I), 2,2-dibromo-N-((2S,3R)-1,3-dihydroxyoctadecan-2-il)acetamide or 2-bromo-N-((2S,3R)-1,3-dihydroxyoctadecan-2-il)acetamide in the treatment or prevention of a disease associated with cellular hyperproliferation, either alone or in combination.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad de Barcelona (España)A1 Solicitud de patente con informe sobre el estado de la técnic