40 Years of RAS—A Historic Overview

[ES]Han pasado más de cuarenta años desde el aislamiento del primer oncogén humano (HRAS), un hito crucial en la investigación del cáncer que fue posible gracias a los esfuerzos combinados de unos pocos grupos de investigación seleccionados a principios de los años ochenta. Esos descubrimientos iniciales condujeron a un salto cuantitativo en nuestra comprensión de la biología del cáncer y marcaron el inicio del campo de la oncología molecular. Las siguientes cuatro décadas de investigación de RAS han producido una enorme cantidad de nuevos conocimientos sobre la familia de pequeñas GTPasas RAS, incluido cómo regulan las vías de señalización que controlan muchos procesos fisiológicos celulares, o cómo las mutaciones oncogénicas desencadenan condiciones patológicas, incluidos síndromes del desarrollo o muchos tipos de cáncer. Sin embargo, a pesar del amplio conjunto de conocimientos básicos disponibles, todavía faltan tratamientos eficaces específicos para los cánceres provocados por RAS. Con suerte, los avances recientes que implican el descubrimiento de nuevos bolsillos en la superficie de RAS, así como el descubrimiento de pequeñas moléculas inhibitorias altamente específicas capaces de bloquear la interacción de RAS con sus efectores y/o activadores, y que pueden conducir al desarrollo de tratamientos nuevos y eficaces para el cáncer. Esta revisión pretende proporcionar una descripción histórica rápida y resumida de los principales hitos en la investigación de RAS que abarca desde el descubrimiento inicial de los oncogenes RAS virales en tumores de roedores hasta los últimos intentos de atacar los oncogenes RAS en varios cánceres humanos.[EN]It has been over forty years since the isolation of the first human oncogene (HRAS), a crucial milestone in cancer research made possible through the combined efforts of a few selected research groups at the beginning of the 1980s. Those initial discoveries led to a quantitative leap in our understanding of cancer biology and set up the onset of the field of molecular oncology. The following four decades of RAS research have produced a huge pool of new knowledge about the RAS family of small GTPases, including how they regulate signaling pathways controlling many cellular physiological processes, or how oncogenic mutations trigger pathological conditions, including developmental syndromes or many cancer types. However, despite the extensive body of available basic knowledge, specific effective treatments for RAS-driven cancers are still lacking. Hopefully, recent advances involving the discovery of novel pockets on the RAS surface as well as highly specific small-molecule inhibitors able to block its interaction with effectors and/or activators may lead to the development of new, effective treatments for cancer. This review intends to provide a quick, summarized historical overview of the main milestones in RAS research spanning from the initial discovery of the viral RAS oncogenes in rodent tumors to the latest attempts at targeting RAS oncogenes in various human cancers.ISCIII-MCUI (FIS PI19/00934), JCyL (SA264P18-UIC076), Fundación Ramón Areces (CIVP19A5942) e ISCIII-CIBERONC (CB16/12/00352). Cofinanciado con fondos FEDER

Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption

[ES]El consumo de alcohol es un rasgo moderadamente hereditario, pero la base genética en los humanos se desconoce en gran medida, a pesar de su importancia clínica y social. Presentamos un metanálisis de un estudio de asociación de todo el genoma con aproximadamente 2,5 millones de SNP genotipados o imputados directamente con consumo de alcohol (gramos por día por kilogramo de peso corporal) entre 12 muestras poblacionales de ascendencia europea, que comprenden 26.316 individuos, con posterior genotipado de comprobación en 21.185 personas más. El SNP rs6943555 en el gen candidato 2 de susceptibilidad al autismo (AUTS2) se asoció con el consumo de alcohol con significación en todo el genoma (P = 4 × 10−8 a P = 4 × 10−9). Encontramos una expresión de AUTS2 específica de genotipo en 96 muestras de corteza prefrontal humana (P = 0,026) y diferencias significativas (P <0,017) en la expresión de AUTS2 en extractos de cerebro completo de ratones seleccionados por diferencias en el consumo voluntario de alcohol. La regulación negativa de un homólogo de AUTS2 provocó una reducción de la sensibilidad al alcohol en Drosophila (P <0,001). Nuestro hallazgo de un regulador del consumo de alcohol añade conocimiento a nuestra comprensión de los mecanismos genéticos que influyen en el comportamiento de consumo de alcohol.[EN]Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10−8 to P = 4 × 10−9 ). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Downregulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior.Este trabajo fue apoyado por subvenciones del ISCIII (FIS PS09/01979) y JCyL (SA044A08 y GR93), así como el apoyo institucional de la RTICC (RD06/0020/000)

Genetic analysis of Ras signalling pathways in cell proliferation, migration and survival

We have used mouse embryonic fibroblasts (MEFs) devoid of Ras proteins to illustrate that they are essential for proliferation and migration, but not for survival, at least in these cells. These properties are unique to the Ras subfamily of proteins because ectopic expression of other Ras-like small GTPases, even when constitutively active, could not compensate for the absence of Ras proteins. Only constitutive activation of components of the Raf/Mek/Erk pathway was sufficient to sustain normal proliferation and migration of MEFs devoid of Ras proteins. Activation of the phosphatidylinositol 3-kinase (PI3K)/PTEN/Akt and Ral guanine exchange factor (RalGEF)/Ral pathways, either alone or in combination, failed to induce proliferation or migration of Rasless cells, although they cooperated with Raf/Mek/Erk signalling to reproduce the full response mediated by Ras signalling. In contrast to current hypotheses, Ras signalling did not induce proliferation by inducing expression of D-type Cyclins. Rasless MEFs had normal levels of Cyclin D1/Cdk4 and Cyclin E/Cdk2. However, these complexes were inactive. Inactivation of the pocket proteins or knock down of pRb relieved MEFs from their dependence on Ras signalling to proliferate.This work was supported by grants from the Ministry of Science and Innovation (MICINN) (SAF2006-11773 and Consolider-Ingenio 2010, CSD2007-00017), the Comunidad Autónoma de Madrid (S-BIO-0283-2006) and the 7th Framework Programme (CHEMORES LSHG-CT-2007-037665) to MB, by grants from the Junta de Castilla y León (SA044A08 and GR93) and the Instituto de Salud Carlos III (ISCIII) (FIS PI021570) to ES and by institutional support from the RTICC (RD06/0020/000) and Acción Transversal en Cáncer 2008 from the ISCIII (also to ES). MD was supported by postdoctoral fellowships from the Ernst Schering Foundation and the Deutsche Forschungsgemeinschaft. EYMS was a recipient of a CJ Martin postdoctoral fellowship from the National Health and Medical Research Council (Australia).Peer Reviewe

GRF2 Is crucial for cone photoreceptor viability and ribbon synapse formation in the mouse retina

[ES]Utilizando ratones knockout para GRF1/2 constitutivos, demostramos previamente que GRF2 es un regulador clave de la migración nuclear en los fotorreceptores de los conos de la retina. Para evaluar la relevancia funcional de ese proceso celular para dos objetivos de la actividad GEF de GRF2 (RAC1 y CDC42), aquí comparamos los fenotipos retinianos estructurales y funcionales resultantes de la eliminación condicional de RAC1 o CDC42 en los fotorreceptores de tipo cono en un fondo GRF2 KO constitutivo comparando con Ratones GRF2 WT. Observamos que la eliminación individual de RAC1 no causó ningún cambio morfológico o fisiológico obvio en las retinas de los ratones GRF2 WT, y tampoco modificó las alteraciones fenotípicas descritas previamente en la capa de fotorreceptores retinianos de los ratones GRF2 KO. Por el contrario, la ablación de CDC42 en los conos de ratones GRF2 WT resultó en claras alteraciones del movimiento nuclear que, a diferencia de las observadas en las retinas GRF2KO, no estuvieron acompañadas de defectos electrofisiológicos ni de una degeneración lenta y progresiva de los conos. Por otro lado, la eliminación concomitante de GRF2 y CDC42 en los conos resultó, sorprendente, en un patrón normalizado de posicionamiento/movimiento nuclear, similar al observado fisiológicamente en ratones GRF2 WT, así como alteraciones electrofisiológicas y una muerte celular de los conos más acusadas que los registrados previamente en células de cono GRF2KO individuales. Curiosamente, las mayores tasas de apoptosis en los conos observadas en las retinas GRF2 KO simple y GRF2 KO/CDC42 KO se correlacionaron con la detección por microscopía electrónica de alteraciones ultraestructurales importantes (aplanamiento) de sus sinapsis en cinta en las retinas, que no se observaron en retinas CDC42 KO. Nuestras observaciones identifican a GRF2 y CDC42 (pero no a RAC1) como reguladores clave de los procesos retinianos que controlan el posicionamiento y la supervivencia nuclear de los fotorreceptores de cono, y respaldan la noción de mutaciones de pérdida de función de GRF2 como posibles impulsores de las distrofias retinianas de los conos.[EN]Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuclear migration in retinal cone photoreceptors. To evaluate the functional relevance of that cellular process for two putative targets of the GEF activity of GRF2 (RAC1 and CDC42), here we compared the structural and functional retinal phenotypes resulting from conditional targeting of RAC1 or CDC42 in the cone photoreceptors of constitutive GRF2KO and GRF2WT mice. We observed that single RAC1 disruption did not cause any obvious morphological or physiological changes in the retinas of GRF2WT mice, and did not modify either the phenotypic alterations previously described in the retinal photoreceptor layer of GRF2KO mice. In contrast, the single ablation of CDC42 in the cone photoreceptors of GRF2WT mice resulted in clear alterations of nuclear movement that, unlike those of the GRF2KO retinas, were not accompanied by electrophysiological defects or slow, progressive cone cell degeneration. On the other hand, the concomitant disruption of GRF2 and CDC42 in the cone photoreceptors resulted, somewhat surprisingly, in a normalized pattern of nuclear positioning/movement, similar to that physiologically observed in GRF2WT mice, along with worsened patterns of electrophysiological responses and faster rates of cell death/disappearance than those previously recorded in single GRF2KO cone cells. Interestingly, the increased rates of cone cell apoptosis/death observed in single GRF2KO and double-knockout GRF2KO/CDC42KO retinas correlated with the electron microscopic detection of significant ultrastructural alterations (flattening) of their retinal ribbon synapses that were not otherwise observed at all in single-knockout CDC42KO retinas. Our observations identify GRF2 and CDC42 (but not RAC1) as key regulators of retinal processes controlling cone photoreceptor nuclear positioning and survival, and support the notion of GRF2 loss-of-function mutations as potential drivers of cone retinal dystrophies.ISCIII-MCUI (FIS PI19/00934 y FIS-PI22/01538), JCyL (SA264P18-UIC 076), Fundación Ramón Areces (Madrid, CIVP19A5942) y Fundación Memoria Samuel Solorzano Barruso (FS/22-2019, FS/32-2020 and FS/14-2021), e ISCIII-CIBERONC (grupo CB16/12/00352). Investigación co-financiada con fondos FEDER

Targeted genomic disruption of H-ras and N-ras has no effect on early renal changes after unilateral ureteral ligation

[Purpose]: To assess the contribution of two different Ras monomeric GTPases isoforms H- and N-Ras in the early changes associated to obstructive nephropathy induced by unilateral ureteral obstruction (UUO). [Methods]: UUO was performed in N-ras (N-ras -/- ) and H-ras (H-ras -/- ) knock-out mice and control (H-ras +/+ /N-ras +/+ ) mice of C57Bl/6 background. Fibronectin, α-smooth muscle actin, cleaved caspase-3, ki-67, Ras-GTP, pERK, and pAkt expression was analyzed by western blot and/or immunohistochemistry. Ras isoforms activation and caspase activity were determined by both western blot and ELISA. [Results]: Three days after UUO, obstructed (O) kidneys of H-ras -/-, N-ras -/- and H-ras +/+ /N-ras +/+ mice showed no significant differences in activated total ras, pERK1/2, pAkt, total Akt levels, fibronectin, α-SMA expression, cell proliferation, and activated caspase-3. The morphological alterations in the O kidneys, revealed by histological and immunohistochemical studies, were also similar in H-ras -/-, N-ras -/-, and H-ras +/+ /N-ras +/+ mice. [Conclusions]: These data suggest that the activation of H-ras and N-ras isoforms does not play a major role in the early renal damage induced by UUO. © 2009 Springer-Verlag.This study was supported by grants from Spanish Ministerio de Ciencia y Tecnología (BFU2004-00285/BFI and SAF 2003-04177), Instituto de Salud Carlos III (RD06/0016/013: RedinRen) and Junta de Castilla y León (SA 001/C05).Peer Reviewe

Ras-Guanine Nucleotide Exchange Factor Sos2 Is Dispensable for Mouse Growth and Development

The mammalian sos1 and sos2 genes encode highly homologous members of the Son-of-sevenless family of guanine nucleotide exchange factors. They are ubiquitously expressed and play key roles in transmission of signals initiated by surface protein tyrosine kinases that are transduced into the cell through the action of membrane-associated Ras proteins. Recent reports showed that targeted disruption of the sos1 locus results in embryonic lethality. To gain insight into the in vivo function of sos2, we disrupted its catalytic CDC25-H domain by means of gene targeting techniques. Mating among heterozygous sos2+/− mice produced viable sos2−/− offspring with a normal Mendelian pattern of inheritance, indicating that the loss of sos2 does not interfere with embryo viability in the uterus. Adult homozygous mutant sos2−/− mice reached sexual maturity at the same age as their wild-type littermates, and both male and female null mutants were fertile. Histopathological analysis showed no observable differences between mutant and wild-type mice. Our results show that unlike the case for sos1, sos2 gene function is dispensable for normal mouse development, growth, and fertility.Peer reviewe

PO-187 Defective liver regeneration ability of Sos1-KO and Sos1/2-DKO, but not Sos2-KO mice

[ES]La pérdida combinada de Sos1 y Sos2 en ratones DKO resultó en niveles marcadamente reducidos de proteína sérica total y aumento de los niveles séricos de lactato deshidrogenasa, creatinina quinasa y otras enzimas hepáticas, lo que sugiere una insuficiencia hepática sustancial en estos animales. El análisis histológico de los animales DKO mostró una rápida degeneración estructural del hígado acompañada de mayores niveles de estrés oxidativo en los lóbulos hepáticos. También observamos una marcada distensión de la vesícula biliar, quizás relacionada con una disminución de los niveles de CCK en las células de la mucosa intestinal. La dilatación de la vesícula biliar junto con la disminución observada de los triglicéridos séricos y de la grasa en todos los órganos analizados apuntan a un deterioro significativo del metabolismo lipídico en ratones Sos1/2-DKO. Finalmente, nuestros estudios con hepatectomía parcial en ratones Sos KO individuales mostraron que la regeneración hepática depende críticamente de la presencia de Sos1, mientras que Sos2 parece ser prescindible para este proceso de recuperación de órganos después de un daño hepático.AECC, PI16/0213, PI19/01083, CB16/12/00352 (ISCIII) and SA043U16 (CyL-FEDER

Evolution over Time of Ventilatory Management and Outcome of Patients with Neurologic Disease∗

OBJECTIVES: To describe the changes in ventilator management over time in patients with neurologic disease at ICU admission and to estimate factors associated with 28-day hospital mortality. DESIGN: Secondary analysis of three prospective, observational, multicenter studies. SETTING: Cohort studies conducted in 2004, 2010, and 2016. PATIENTS: Adult patients who received mechanical ventilation for more than 12 hours. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Among the 20,929 patients enrolled, we included 4,152 (20%) mechanically ventilated patients due to different neurologic diseases. Hemorrhagic stroke and brain trauma were the most common pathologies associated with the need for mechanical ventilation. Although volume-cycled ventilation remained the preferred ventilation mode, there was a significant (p &lt; 0.001) increment in the use of pressure support ventilation. The proportion of patients receiving a protective lung ventilation strategy was increased over time: 47% in 2004, 63% in 2010, and 65% in 2016 (p &lt; 0.001), as well as the duration of protective ventilation strategies: 406 days per 1,000 mechanical ventilation days in 2004, 523 days per 1,000 mechanical ventilation days in 2010, and 585 days per 1,000 mechanical ventilation days in 2016 (p &lt; 0.001). There were no differences in the length of stay in the ICU, mortality in the ICU, and mortality in hospital from 2004 to 2016. Independent risk factors for 28-day mortality were age greater than 75 years, Simplified Acute Physiology Score II greater than 50, the occurrence of organ dysfunction within first 48 hours after brain injury, and specific neurologic diseases such as hemorrhagic stroke, ischemic stroke, and brain trauma. CONCLUSIONS: More lung-protective ventilatory strategies have been implemented over years in neurologic patients with no effect on pulmonary complications or on survival. We found several prognostic factors on mortality such as advanced age, the severity of the disease, organ dysfunctions, and the etiology of neurologic disease