http://hdl.handle.net/10366/4578 Wed, 22 Apr 2026 22:12:40 GMT 2026-04-22T22:12:40Z http://hdl.handle.net/10366/162496 [EN]Purpose: The search for new drugs that control the continuous relapses of multiple myeloma is still required. Here, we report for the first time the potent antimyeloma activity of amiloride, an old potassium-sparing diuretic approved for the treatment of hypertension and edema due to heart failure.Experimental Design: Myeloma cell lines and primary samples were used to evaluate cytotoxicity of amiloride. In vivo studies were carried out in a xenograft mouse model. The mechanisms of action were investigated using RNA-Seq experiments, qRT-PCR, immunoblotting, and immunofluorescence assays.Results: Amiloride-induced apoptosis was observed in a broad panel of multiple myeloma cell lines and in a xenograft mouse model. Moreover, amiloride also had a synergistic effect when combined with dexamethasone, melphalan, lenalidomide, and pomalidomide. RNA-Seq experiments showed that amiloride not only significantly altered the level of transcript isoforms and alternative splicing events, but also deregulated the spliceosomal machinery. In addition, disruption of the splicing machinery in immunofluorescence studies was associated with the inhibition of myeloma cell viability after amiloride exposure. Although amiloride was able to induce apoptosis in myeloma cells lacking p53 expression, activation of p53 signaling was observed in wild-type and mutated TP53 cells after amiloride exposure. On the other hand, we did not find a significant systemic toxicity in mice treated with amiloride.Conclusions: Overall, our results demonstrate the antimyeloma activity of amiloride and provide a mechanistic rationale for its use as an alternative treatment option for relapsed multiple myeloma patients, especially those with 17p deletion or TP53 mutations that are resistant to current therapies. Sun, 01 Jan 2017 00:00:00 GMT http://hdl.handle.net/10366/162496 2017-01-01T00:00:00Z http://hdl.handle.net/10366/162485 [EN]The development of resistance to therapy is unavoidable in the history of multiple myeloma patients. Therefore, the study of its characteristics and mechanisms is critical in the search for novel therapeutic approaches to overcome it. This effort is hampered by the absence of appropriate preclinical models, especially those mimicking acquired resistance. Here we present an in vivo model of acquired resistance based on the continuous treatment of mice bearing subcutaneous MM1S plasmacytomas. Xenografts acquired resistance to two generations of immunomodulatory drugs (IMiDs; lenalidomide and pomalidomide) in combination with dexamethasone, that was reversible after a wash-out period. Furthermore, lenalidomide-dexamethasone (LD) or pomalidomide-dexamethasone (PD) did not display cross-resistance, which could be due to the differential requirements of the key target Cereblon and its substrates Aiolos and Ikaros observed in cells resistant to each combination. Differential gene expression profiles of LD and PD could also explain the absence of cross-resistance. Onset of resistance to both combinations was accompanied by upregulation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway and addition of selumetinib, a small-molecule MEK inhibitor, could resensitize resistant cells. Our results provide insights into the mechanisms of acquired resistance to LD and PD combinations and offer possible therapeutic approaches to addressing IMiD resistance in the clinic. Wed, 01 Jan 2014 00:00:00 GMT http://hdl.handle.net/10366/162485 2014-01-01T00:00:00Z http://hdl.handle.net/10366/162473 [EN]Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy. Wed, 01 Jan 2014 00:00:00 GMT http://hdl.handle.net/10366/162473 2014-01-01T00:00:00Z http://hdl.handle.net/10366/162464 [EN]Despite recent advances in the treatment of multiple myeloma (MM), it remains an incurable disease potentially due to the presence of resistant myeloma cancer stem cells (MM-CSC). Although the presence of clonogenic cells in MM was described three decades ago, the phenotype of MM-CSC is still controversial, especially with respect to the expression of syndecan-1 (CD138). Here, we demonstrate the presence of two subpopulations--CD138++ (95-99%) and CD138low (1-5%)--in eight MM cell lines. To find out possible stem-cell-like features, we have phenotypically, genomic and functionally characterized the two subpopulations. Our results show that the minor CD138low subpopulation is morphologically identical to the CD138++ fraction and does not represent a more immature B-cell compartment (with lack of CD19, CD20 and CD27 expression). Moreover, both subpopulations have similar gene expression and genomic profiles. Importantly, both CD138++ and CD138low subpopulations have similar sensitivity to bortezomib, melphalan and doxorubicin. Finally, serial engraftment in CB17-SCID mice shows that CD138++ as well as CD138low cells have self-renewal potential and they are phenotypically interconvertible. Overall, our results differ from previously published data in MM cell lines which attribute a B-cell phenotype to MM-CSC. Future characterization of clonal plasma cell subpopulations in MM patients' samples will guarantee the discovery of more reliable markers able to discriminate true clonogenic myeloma cells. Fri, 21 Mar 2014 00:00:00 GMT http://hdl.handle.net/10366/162464 2014-03-21T00:00:00Z http://hdl.handle.net/10366/161161 [SPA] C3G es un factor de intercambio de nucleótidos de guanina para Rap1 que controla la activación y agregación plaquetaria, así como la liberación del contenido de los gránulos alfa. La expresión transgénica de C3G en plaquetas produce un secretoma netamente proangiogénico mediante la retención de trombospondina-1. En un contexto fisiológico, C3G también promueve la maduración de los megacariocitos y la formación de proplaquetas, sin afectar la producción de plaquetas maduras. El objetivo de este trabajo fue investigar si C3G está implicado en la megacariopoyesis patológica, así como su papel específico en la angiogénesis y metástasis mediadas por plaquetas. Utilizando modelos de ratones con eliminación o sobreexpresión específica de C3G en megacariocitos, encontramos que tanto la sobreexpresión como la eliminación de C3G promovieron la angiogénesis mediada por plaquetas, inducida por la implantación de células tumorales o por isquemia del miembro posterior, mediante la liberación diferencial de factores proangiogénicos y antiangiogénicos. La eliminación de C3G también produjo un mayor reclutamiento de hemangiocitos provenientes de la médula ósea. Por otra parte, la ausencia de C3G incrementó la producción de plaquetas inducida por trombopoyetina (TPO), asociada con niveles plasmáticos reducidos de TPO. Asimismo, tras la depleción de plaquetas inducida por 5-fluorouracilo, los ratones knockout para C3G mostraron una recuperación mas lenta de los niveles homeostáticos de plaquetas, indicando una alteración en el proceso de renovación plaquetaria. Mecanísticamente, C3G promueve la ubiquitinación del receptor c-Mpl mediante la inducción de la fosforilación de c-Cbl mediada por Src, y participa en la degradación de c-Mpl a través de los sistemas proteasoma y lisosoma, afectando la internalización de TPO. Nuestros resultados también revelaron un papel positivo de C3G plaquetario en la agregación plaquetaria inducida por células tumorales, lo que facilitó la adhesión y el establecimiento de las células metastásicas. En conjunto, estos hallazgos revelan que C3G desempeña un papel crucial en la angiogénesis y la metástasis mediadas por plaquetas, así como en la modulación de los niveles de plaquetas en respuesta a estímulos patológicos.  Sat, 01 Jan 2022 00:00:00 GMT http://hdl.handle.net/10366/161161 2022-01-01T00:00:00Z http://hdl.handle.net/10366/161155 [SPA] Datos previos indicaron que la isoforma principal de C3G (RapGEF1) está altamente expresada en células progenitoras hepáticas (o células ovales) en comparación con hepatocitos maduros adultos, lo que sugiere que podría desempeñar un papel importante en la biología de las células ovales. En este trabajo hemos explorado la función de C3G en la regulación de las propiedades de las células ovales mediante el silenciamiento permanente del gen utilizando shRNAs. La reducción de C3G aumentó la capacidad migratoria e invasiva de las células ovales al promover una transición epitelial a mesenquimal (EMT) parcial. Esto probablemente se deba a un incremento de la expresión de ARNm de los factores de transcripción inductores de EMT, Snail1, Zeb1 y Zeb2, en células ovales con silenciación de C3G. Esta EMT está asociada con una mayor expresión de los marcadores de pluripotencia CD133 y CD44. Además, la disminución de C3G incrementó la capacidad clonogénica de las células ovales al potenciar su dispersión celular. Sin embargo, el silenciamiento de C3G no afectó la diferenciación de las células ovales hacia la línea de hepatocitos. Los estudios mecanísticos revelaron que la señalización de HGF/MET y su actividad proinvasiva estaban alteradas en células ovales con niveles bajos de C3G, mientras que la señalización de TGF-β estaba incrementada. En conjunto, estos datos sugieren que los niveles de C3G deben estar estrictamente regulados para asegurar la reparación hepática en enfermedades crónicas del hígado, como la esteatohepatitis no alcohólica. Así, niveles reducidos de C3G podrían facilitar la expansión de las células ovales después del pico de proliferación al aumentar su migración. Sat, 01 Jan 2022 00:00:00 GMT http://hdl.handle.net/10366/161155 2022-01-01T00:00:00Z http://hdl.handle.net/10366/161151 [EN] C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.; [ES] C3G es un factor de intercambio de nucleótidos de guanina (GEF) para Rap1 que desempeña un papel crucial en procesos mediados por plaquetas como la angiogénesis, el crecimiento tumoral y la metástasis, modulando el secretoma plaquetario. En este estudio, exploramos los mecanismos mediante los cuales C3G regula la secreción plaquetaria. Para ello, utilizamos modelos animales con sobreexpresión o eliminación de C3G en plaquetas, así como clones de células PC12 que expresan mutantes de C3G. Descubrimos que C3G regula específicamente la secreción de los gránulos alfa plaquetarios a través de PKCδ, pero no afecta a los gránulos densos ni a los lisosomas. C3G activa a la GTPasa RalA mediante un mecanismo dependiente de su actividad GEF, facilitando el acoplamiento de vesículas, pero interfiriendo con la formación del complejo trans-SNARE, lo que restringe la fusión de vesículas. Además, C3G promueve la formación de lamelipodios durante la extensión de plaquetas sobre sustratos específicos al potenciar la polimerización de actina mediante las vías Src y Rac1-Arp2/3, pero no a través de Rap1. Como consecuencia, la eliminación de C3G en plaquetas favorece la exocitosis tipo kiss-and-run. C3G también controla la secreción de gránulos en células PC12, incluido el proceso de formación de poros. Adicionalmente, las plaquetas deficientes en C3G mostraron una menor exposición de fosfatidilserina, lo que resultó en una disminución en la generación de trombina. Esto, junto con una polimerización defectuosa de actina y una extensión sobre sustrato limitada, resulta en una retracción del coágulo deficiente. En resumen, C3G en plaquetas desempeña un doble papel: facilita la extensión de las plaquetas y la retracción del coágulo mediante la promoción de la señalización outside-in, mientras que simultáneamente regula negativamente la secreción de gránulos alfa al restringir la fusión de los gránulos a la membrana. Mon, 01 Jan 2024 00:00:00 GMT http://hdl.handle.net/10366/161151 2024-01-01T00:00:00Z http://hdl.handle.net/10366/161150 [SPA]El glioblastoma (GBM) es el tumor más agresivo del sistema nervioso central (SNC). La actual falta de terapias eficaces hace esencial encontrar nuevas estrategias de tratamiento. C3G, un factor de intercambio de nucleótidos de guanina para algunas proteínas Ras, desempeña un papel dual en el cáncer, pero su función en GBM sigue siendo desconocida. Los análisis de bases de datos revelaron una expresión reducida de ARNm de C3G en muestras de pacientes con GBM. Además, los niveles de proteína C3G también estaban disminuidos en un panel de líneas celulares humanas de GBM en comparación con los astrocitos. Con base en estos hallazgos, hemos caracterizado la función de C3G en GBM utilizando líneas celulares humanas de GBM in vitro e in vivo. La reducción de C3G promovió la adquisición de un fenotipo más mesenquimal que aumentó la capacidad migratoria e invasiva de las células de GBM. Esto facilitó la formación de focos en ensayos de crecimiento dependiente e independiente de anclaje, así como la generación de tumores de mayor tamaño en modelos de xenoinjertos y en la membrana corioalantoidea (CAM) de pollo, aunque con una menor densidad celular, debido a una menor proliferación. Mecanísticamente, la reducción de C3G afecta la señalización de EGFR al disminuir el contenido de EGFR en la superficie celular debido a la inhibición de su reciclaje, mientras que aumenta la activación de otros receptores tirosina quinasas (RTKs) que podrían promover la invasión. En particular, el FGF2, probablemente actuando a través de su receptor FGFR1, promovió la invasión de células de GBM con silenciación de C3G. Además, las ERK median esta capacidad invasiva, tanto en respuesta a FGF2 como a quimioatracción inducida por suero. En conclusión, nuestros datos muestran la dependencia distintiva de los tumores de GBM en C3G para la señalización de EGF/EGFR frente a otros RTKs. Esto sugiere que la evaluación de los niveles de C3G podría discriminar a los pacientes con GBM que responden a diferentes protocolos de inhibición de RTK. Por lo tanto, los pacientes con baja expresión de C3G podrían no responder a los inhibidores de EGFR. Fri, 01 Jan 2021 00:00:00 GMT http://hdl.handle.net/10366/161150 2021-01-01T00:00:00Z http://hdl.handle.net/10366/161149 [SPA]C3G es un factor de intercambio de nucleótidos de guanina (GEF, por sus siglas en inglés) para las GTPasas Rap, entre las cuales la isoforma Rap1b es una proteína esencial en la biología de las plaquetas. Utilizando modelos de ratones transgénicos con sobreexpresión específica en plaquetas de C3G (tgC3G) o del mutante C3GΔCat (tgC3GΔCat, carente del dominio catalítico), así como un modelo de ratón con eliminación específica de C3G en plaquetas (C3G-KO), en este trabajo hemos demostrado que C3G contribuye a la activación y agregación plaquetaria, tanto in vitro como in vivo, a través de la activación de Rap1b. Además, hemos explorado la contribución de la vía C3G-Rap1b a las rutas de señalización plaquetarias desencadenadas por trombina, PMA o ADP en el modelo de ratón transgénico referido (tgC3G) mediante el uso de una batería de inhibidores específicos. Nuestros resultados indican que la proteína C3G plaquetaria es fosforilada en Tyr504 mediante un mecanismo que involucra a la ruta de PKC-Src. Esta fosforilación está regulada positivamente por las ERK/MAPK a través de la inhibición de la fosfatasa de tirosina Shp2. Además, C3G participa en la vía ADP-P2Y12-PI3K-Rap1b y es un mediador de la actividad de la ruta trombina-TXA2. Sin embargo, C3G inhibe la síntesis de TXA2 mediante la regulación de cPLA2. En conjunto, nuestros datos revelan un papel clave de C3G en las principales vías que conducen a la activación y agregación plaquetaria a través de la regulación de Rap1b. Wed, 01 Jan 2020 00:00:00 GMT http://hdl.handle.net/10366/161149 2020-01-01T00:00:00Z http://hdl.handle.net/10366/156737 [EN]The R-RAS2 GTP hydrolase (GTPase) (also known as TC21) has been traditionally considered quite similar to classical RAS proteins at the regulatory and signaling levels. Recently, a long-tail hotspot mutation targeting the R-RAS2/TC21 Gln72 residue (Q72L) was identified as a potent oncogenic driver. Additional point mutations were also found in other tumors at low frequencies. Despite this, little information is available regarding the transforming role of these mutant versions and their relevance for the tumorigenic properties of already-transformed cancer cells. Here, we report that many of the RRAS2 mutations found in human cancers are highly transforming when expressed in immortalized cell lines. Moreover, the expression of endogenous R-RAS2Q72L is important for maintaining optimal levels of PI3K and ERK activities as well as for the adhesion, invasiveness, proliferation, and mitochondrial respiration of ovarian and breast cancer cell lines. Endogenous R-RAS2Q72L also regulates gene expression programs linked to both cell adhesion and inflammatory/immune-related responses. Endogenous R-RAS2Q72L is also quite relevant for the in vivo tumorigenic activity of these cells. This dependency is observed even though these cancer cell lines bear concurrent gain-of-function mutations in genes encoding RAS signaling elements. Finally, we show that endogenous R-RAS2, unlike the case of classical RAS proteins, specifically localizes in focal adhesions. Collectively, these results indicate that gain-of-function mutations of R-RAS2/TC21 play roles in tumor initiation and maintenance that are not fully redundant with those regulated by classical RAS oncoproteins. Sun, 01 Jan 2023 00:00:00 GMT http://hdl.handle.net/10366/156737 2023-01-01T00:00:00Z http://hdl.handle.net/10366/156736 [EN]The impact of genetic ablation of SOS1 or SOS2 is evaluated in a murine model of KRASG12D-driven lung adenocarcinoma (LUAD). SOS2 ablation shows some protection during early stages but only SOS1 ablation causes significant, specific long term increase of survival/lifespan of the KRASG12D mice associated to markedly reduced tumor burden and reduced populations of cancer-associated fibroblasts, macrophages and T-lymphocytes in the lung tumor microenvironment (TME). SOS1 ablation also causes specific shrinkage and regression of LUAD tumoral masses and components of the TME in pre-established KRASG12D LUAD tumors. The critical requirement of SOS1 for KRASG12D-driven LUAD is further confirmed by means of intravenous tail injection of KRASG12D tumor cells into SOS1KO/KRASWT mice, or of SOS1-less, KRASG12D tumor cells into wildtype mice. In silico analyses of human lung cancer databases support also the dominant role of SOS1 regarding tumor development and survival in LUAD patients. Our data indicate that SOS1 is critically required for development of KRASG12D-driven LUAD and confirm the validity of this RAS-GEF activator as an actionable therapeutic target in KRAS mutant LUAD. Wed, 20 Sep 2023 00:00:00 GMT http://hdl.handle.net/10366/156736 2023-09-20T00:00:00Z http://hdl.handle.net/10366/156634 [EN]The isolation of the first human oncogene (HRAS), a critical breakthrough in cancer research, has occurred over forty years ago, and the identification of new pathogenic oncogenes has continuously grown since [...]. Thu, 13 Apr 2023 00:00:00 GMT http://hdl.handle.net/10366/156634 2023-04-13T00:00:00Z http://hdl.handle.net/10366/154481 [EN] The aim of this study was to assess patterns of CCND3 gene amplification in bladder cancer and correlate gene status with recurrence-free and progression-free survival. A sequential cohort series of 102 primary bladder tumor samples in which there was enough tissue material to assess CCND3 gene status by fluorescent in situ hybridization (FISH) was the study group. CCND3 gene FISH amplification present in 31.4 percent of bladder carcinomas, was related to tumor progression (p=0.021) and lower time to progression (mean+-SD; 25.75+-15.25 months) as compared to 33.29+-11.0 months in the CCND3 not amplified group (p=0.05). By immunohistochemistry, Cyclin D3 labeling index was higher in the CCND3 amplified group (mean+-SD, 76.69+-27.51) than in not amplified (mean+-SD, 21.57+-7.02) (p less than 0.0001). The univariate survival analysis showed CCND3 gene amplification to be associated to a shorter progression-free survival (p=0.020) together with WHO histological grade (p=0.001) and pT stage category (p less than 0.0001). Cox's regression analysis selected CCND3 amplification as an independent predictor of progression-free survival (p= 0.030, RR3.561, 95 percent CI 1.128-11.236) together with pT category (p less than 0.0001, RR5.834, 95 percent CI 2.364-14.395). Our FISH analysis suggests that CCND3 gene amplification is a marker of aggressiveness and might be a predictor of tumor progression in bladder urothelial carcinoma. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10366/154481 2013-01-01T00:00:00Z http://hdl.handle.net/10366/154480 [EN]Purpose: Goal of this study was to identify mechanisms that limit efficacy of trabectedin (ET-743, Yondelis) in Ewing sarcoma (EWS), so as to develop a clinical applicable combination therapy. Experimental Design: By chromatin immunoprecipitation, we analyzed EWS-FLI1 binding to the promoters of several target genes, such as TGFβR2, CD99, insulin-like growth factor receptor 1 (IGF1R), and IGF1, both in vitro and in xenografts treated with trabectedin or doxorubicin. Combined therapy with trabectedin and anti-IGF1R agents (AVE1642 HAb; OSI-906) was tested in vitro and in xenografts. Results: We confirm that both trabectedin and doxorubicin were able to strongly reduce EWS-FLI1 (both type I and type II) binding to two representative target genes ( TGFβR2 and CD99), both in vitro and in xenografts. However, trabectedin, but not doxorubicin, was also able to increase the occupancy of EWS-FLI1 to IGF1R promoters, leading to IGF1R upregulation. Inhibition of IGF1R either by the specific AVE1642 human antibody or by the dual IGF1R/insulin receptor inhibitor OSI-906 (Linsitinib) greatly potentiate the efficacy of trabectedin in the 13 EWS cell lines here considered as well as in TC-71 and 6647 xenografts. Combined therapy induced synergistic cytotoxic effects. Trabectedin and OSI-906 deliver complementary messages that likely converge on DNA-damage response and repair pathways. Conclusions: Weshowed that trabectedin may not only inhibit but also enhance the binding of EWS-FLI1 to certain target genes, leading to upregulation of IGF1R. We here provide the rationale for combining trabectedin to anti-IGF1R inhibitors. Thu, 01 Jan 2015 00:00:00 GMT http://hdl.handle.net/10366/154480 2015-01-01T00:00:00Z http://hdl.handle.net/10366/154479 [EN]Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5′aza-2-deoxycitidine and/or trichostatin. A restored CaSR expression in MYCN-amplified cell lines. Following 5′aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10366/154479 2013-01-01T00:00:00Z http://hdl.handle.net/10366/154453 [EN] Ewing Sarcoma (EWS) is a mesenchymal-derived tumor that generally arises in bone and soft tissue. Intensive research regarding the pathogenesis of EWS has been insufficient to pinpoint the early events of Ewing sarcomagenesis. However, the Mesenchymal Stem Cell (MSC) is currently accepted as the most probable cell of origin. Materials and Methods: In an initial study regarding a deep characterization of MSC obtained specifically from EWS patients (MSC-P), we compared them with MSC derived from healthy donors (MSC-HD) and EWS cell lines. We evaluated the presence of the EWS-FLI1 gene fusion and EWSR1 gene rearrangements in MSC-P. The presence of the EWS transcript was confirmed by q-RT-PCR. In order to determine early events possibly involved in malignant transformation, we used a multiparameter quantitative strategy that included both MSC immunophenotypic negative/positive markers, and EWS intrinsic phenotypical features. Markers CD105, CD90, CD34 and CD45 were confirmed in EWS samples. Results: We determined that MSC-P lack the most prevalent gene fusion, EWSR1-FLI1 as well as EWSR1 gene rearrangements. Our study also revealed that MSC-P are more alike to MSC-HD than to EWS cells. Nonetheless, we also observed that EWS cells had a few overlapping features with MSC. As a relevant example, also MSC showed CD99 expression, hallmark of EWS diagnosis. However, we observed that, in contrast to EWS cells, MSC were not sensitive to the inhibition of CD99. Conclusions: In conclusion, our results suggest that MSC from EWS patients behave like MSC-HD and are phenotypically different from EWS cells, thus raising important questions regarding MSC role in sarcomagenesis. © 2014 Amaral et al. Wed, 01 Jan 2014 00:00:00 GMT http://hdl.handle.net/10366/154453 2014-01-01T00:00:00Z