Eugenio Santos - Academia.edu (original) (raw)

Papers by Eugenio Santos

Research Square (Research Square), Dec 2, 2022

BMC Genomics, Oct 25, 2013

Background: 4-Hydroxy-tamoxifen (4OHT) triggers Cre-mediated K-Ras removal in [H-Ras −/− ;N-Ras −... more Background: 4-Hydroxy-tamoxifen (4OHT) triggers Cre-mediated K-Ras removal in [H-Ras −/− ;N-Ras −/− ;K-Ras lox/lox ; RERT ert/ert ] fibroblasts, generating growth-arrested "Rasless" MEFs which are able to recover their proliferative ability after ectopic expression of Ras oncoproteins or constitutively active BRAF or MEK1. Results: Comparison of the transcriptional profiles of Rasless fibroblasts with those of MEFs lacking only H-Ras and N-Ras identified a series of differentially expressed mRNAs and microRNAs specifically linked to the disappearance of K-Ras from these cells. The rescue of cell cycle progression in Rasless cells by activated BRAF or MEK1 resulted in the reversal of most such transcriptional mRNA and microRNA alterations. Functional analysis of the differentially expressed mRNAs uncovered a significant enrichment in the components of pathways regulating cell division, DNA/RNA processing and response to DNA damage. Consistent with G1/S blockade, Rasless cells displayed repression of a series of cell cycle-related genes, including Cyclins, Cyclindependent kinases, Myc and E2F transcription targets, and upregulation of Cyclin-dependent kinase inhibitors. The profile of differentially expressed microRNAs included a specific set of oncomiR families and clusters (repressed miR-17~92, miR-106a~363, miR-106b~25, miR-212~132, miR-183~182, and upregulated miR-335) known for their ability to target a specific set of cellular regulators and checkpoint sensors (including Rb, E2F and Cdkns) able to modulate the interplay between the pro-and anti-proliferative or stress-response pathways that are reversibly altered in Rasless cells. Conclusions: Our data suggest that the reversible proliferation phenotype of Rasless cells is the pleiotropic result of interplay among distinct pro-and anti-proliferative, and stress-response pathways modulated by a regulatory circuitry constituted by a specific set of differentially expressed mRNAs and microRNAs and preferentially targeting two cross-talking signalling axes: Myc-Rb-E2F-dependent and Cdkns-p53-dependent pathways.

Small GTPases, May 24, 2016

Despite their homologous structure and central nervous system(CNS) expression patterns, the GRF1 ... more Despite their homologous structure and central nervous system(CNS) expression patterns, the GRF1 and GRF2 guanine nucleotide exchange factors(GEF) appear to play distinct, non-overlapping functions in cellular excitability, synaptic plasticity or neuromodulation. We recently uncovered a new functional role of GRF2 controlling nuclear migration in cone photoreceptors during postnatal neuroepithelial differentiation of the mouse retina. Analyzing GRF2-KO mice, we detected the specific accumulation of abnormally located, "ectopic" cone photoreceptor nuclei in the photoreceptor segment(PS) layer of their retinas. This alteration was accompanied by defective electroretinograms(ERG) indicative of impaired cone-mediated visual function, and accumulation around the "ectopic" nuclei of signaling molecules known to be functionally relevant for intracellular organelle migration, cytoskeletal reorganization or cell polarity establishment including PAR3, PAR6, and the phosphorylated proteins pPAK, pMLC2 and pVASP. We propose a mechanism whereby the absence of a productive functional interaction between GRF2 and its downstream target CDC42 leads to altered formation/structure of PAR-containing, polarity-related macromolecular complexes and abnormal activation of downstream signaling mediated by activated, phosphorylated forms of PAK, VASP and MLC2. As cone photoreceptors are responsible for color vision and visual acuity, these observations are potentially relevant for degenerative diseases of the human retina, harboring almost double number of cones than mice.

Biomedicines, Sep 4, 2021

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

International Journal of Molecular Sciences, Jun 21, 2021

Proceedings of the National Academy of Sciences of the United States of America, Dec 5, 2012

American Journal of Pathology, Apr 1, 2002

Supported by the Spanish Comision Interministerial de Ciencia y Tecnologia (SAF 99/20 and FEDER 1... more Supported by the Spanish Comision Interministerial de Ciencia y Tecnologia (SAF 99/20 and FEDER 1FD97-1678), Associazione Italiana per la Ricerca sul Cancro (AIRC) the National Cancer Institute (grant CA69129 to S. W. M., CORE grant CA21765), and by the American Lebanese Syrian Associated Charities, St. Jude Children's Research Hospital. Genbank accession numbers for the genomic breakpoints of TFG-ALK S, TFG-ALK L and the TFG-ALK XL cDNA are AF390891, AF390892, and AF390893, respectively.

Nature Communications

The impact of genetic ablation of SOS1 or SOS2 is evaluated in a murine model of KRASG12D-driven ... more 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 da...

Cells

Using constitutive GRF1/2 knockout mice, we showed previously that GRF2 is a key regulator of nuc... more 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...

Genes, May 1, 2021

PubMed, Feb 1, 1992

The dbl oncogene was originally identified by transfection of NIH3T3 cells with DNA from a human ... more The dbl oncogene was originally identified by transfection of NIH3T3 cells with DNA from a human diffuse B-cell lymphoma. The dbl oncogene product is a cytoplasmic phosphoprotein distributed between the cytosolic and cytoskeletal matrix-associated membrane fractions. Nucleotide sequence analysis has indicated that the predicted dbl product is very hydrophilic with no detectable similarity to known oncogene products. We have more recently discovered that a region of dbl essential for its transforming activity shows significant sequence similarity to a yeast cell cycle gene, CDC24, which is involved in cell polarity and bud formation in the division cycle of Saccharomyces cerevisiae. This sequence similarity suggests a possible role for dbl in cell division. We report here that the dbl oncogene product is able to induce maturation of Xenopus oocytes. Germinal vesicle breakdown (GVBD) was observed when oocytes were microinjected with the soluble fraction of SF9 insect cells infected with a dbl recombinant baculovirus as well as with the in vitro-transcribed dbl mRNA. Moreover, extracts of oocytes microinjected with dbl mRNA showed activation of H1 histone kinase activity. These findings define a new biologic activity of the dbl product and provide the opportunity to analyse dbl interactions with other components of signaling pathways involved in oocyte maturation.

PubMed, Mar 1, 1996

Insulin-induced differentiation of 3T3 L1 cells can be mimicked by expression of transfected ras ... more Insulin-induced differentiation of 3T3 L1 cells can be mimicked by expression of transfected ras oncogenes but is completely blocked by expression of dominant negative Ras mutants, demonstrating that Ras proteins mediate insulin signaling in these mammalian cells. In contrast, transfection of tyrosine kinase oncogenes including trk and src dose not result in adipocytic differentiation. Transfected raf-1 oncogenes induce partial adipocytic differentiation, while dominant negative raf mutants block partially the insulin-induced differentiation process. Exposure of 3T3 L1 cells to insulin results in formation of the active Ras-GTP complex without GAP tyrosine phosphorylation. Insulin treatment of untransfected 3T3 L1 cells also induced quick activation of cytosolic 42 kDa mitogen-activated protein kinase (MAPK) and a 90 kDa S6 kinase (RSK). The activation of these cytosolic serine-threonine kinases was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with inducible ras oncogenes. Furthermore, insulin-induced activation of MAPK and RSK could be blocked by expression of a transfected, inducible dominant negative Ras mutant (N17). These results indicate that Ras proteins are obligatory intermediates in the activation of cytosolic ERKs by insulin. Insulin treatment of 3T3 L1 cells or expression of transfected ras oncogenes resulted also in hyperphosphorylation of cellular Raf-1. Insulin-induced Raf hyperphosphorylation was inhibited by expression of an inducible, dominant negative Ras mutant (N17). Interestingly, however, expression of transfected raf oncogenes did not induce MAPK or RSK activation, and the insulin-induced activation of these kinases was not blocked by expression of transfected dominant negative raf mutants. These results suggest a functional dissociation between Raf-1 and MAPK/RSK activation in insulin/Ras signaling pathways leading to 3T3 L1 differentiation and are consistent with Raf-1 kinase acting in a parallel pathway to the MAPK/RSK pathway after Ras activation in these cells.

Journal of Biological Chemistry, Oct 1, 1992

Insulin-induced differentiation of 3T3 L1 cells to adipocytes can be mimicked by the expression o... more Insulin-induced differentiation of 3T3 L1 cells to adipocytes can be mimicked by the expression of transfected ras oncogenes but not of the tyrosine-kinase oncogenes src and trk. Expression of two different transfected, dominant inhibitory ras mutants resulted in significant inhibition of insulin-induced differentiation, suggesting that endogenous Ras proteins are mediators of insulin signaling in these cells. Exposure of untransfected 3T3 L1 cells to insulin resulted in significant formation of the active RaseGTP complex, at levels comparable with those resulting from exposure to platelet-derived growth factor. However, whereas exposure of the same cells to platelet-derived growth factor resulted in significant tyrosine phosphorylation of the p2 1'" GTPase-activating protein (GAP), insulintreated cells did not show any detectable levels of de novo GAP tyrosine phosphorylation. Interestingly, insulin caused tyrosine phosphorylation of the p62 polypeptide coprecipitated with GAP by anti-GAP antibodies. Insulin-induced activation of cytosolic MAP kinase activity in untransfected 3T3 L 1 cells was also mimicked by Ras expression (in the absence of insulin) in the same cells transfected with an inducible ras construct. These results confirm that Ras proteins participate in insulin signaling pathways in these mammalian cells and indicate that activation of cytosolic MAP kinases is an early event occurring downstream from Ras activation. However, tyrosine phosphorylation of GAP appears not to be a significant upstream regulatory event in the activation of Ras by insulin. The eukaryotic Ras proteins are thought to play a crucial role in signal transduction pathways mediating proliferation and/or differentiation processes (Barbacid, 1987; Santos and Nebreda, 1989; Hall, 1990; Bourne et al., 1991). Extensive genetic and biochemical evidence indicates that Ras proteins can function downstream of various growth factor-activated receptor tyrosine kinases and upstream of cytosolic serinethreonine kinases (

Science, Aug 2, 1991

pression of CML-BC colony formation by BCR-ABL antisense oligodeoxynudeotides is not yet known; i... more pression of CML-BC colony formation by BCR-ABL antisense oligodeoxynudeotides is not yet known; inhibition of BCR-ABL protein synthesis could remove an essential component in the mitogenic pathway regulated by hematopoietic growth factors as reported (12). Less likely, down-regulation of BCR-ABL expression could determine aberrant differentiation ofblast cells, resulting in a suppression of colony formation. We have provided evidence that leukemia growth can be selectively inhibited by synthetic oligomers targeted against a tumorspecific gene involved in the maintenance of the leukemic phenotype. Synthetic oligodeoxynucleotides complementary to BCR-ABL hybrid genes can be synthesized on an individual basis once the specific BCR-ABL junction is identified; this can be done within a few days of diagnosis and offers the prospect, at least in vitro, of a gene-targeted anti-leukemic therapy.

Virology, Jul 1, 1991

Human immunodeficiency virus type 1 (HIV-l) NEf protein has been reported to share certain bioche... more Human immunodeficiency virus type 1 (HIV-l) NEf protein has been reported to share certain biochemical and structural properties with known oncoproteins like src or ras. To determine whether this is a general property of NEF from various HIV isolates, three different NEF proteins were expressed in Escherichia co/i using a thermoinducible expression system previously exploited to overproduce functionally active p21 ras proteins. ras and NEF proteins expressed in this manner were evaluated in parallel to compare their biochemical and biological properties. In contrast to ras, our NEF protein preparations had no detectable GTP binding but showed autophosphorylation activity when incubated in the presence of either GTP or ATP. This putative autokinase activity was higher in NEF proteins containing threonine at position 15 than in those carrying alanine at that position. Two different NEF genes also failed to induce oncogenic transformation of permanently transfected NIH 3T3 cells under conditions that led to oncogenic transformation using activated ras genes. Also, unlike ras, the NEf gene products failed to induce meiotic maturation when injected into fully grown Xenopus OOCytSS.

Science, Jul 5, 1991

The endogenous mos proto-oncogene product (Mos) is required for meiotic maturation. In Xenops ooc... more The endogenous mos proto-oncogene product (Mos) is required for meiotic maturation. In Xenops oocytes, the ras oncogene product (Ras) can induce meiotic maturation and high levels of M-phase-promoting factor (MPF) independent of endogenous Mos, indicating that a parallel pathway to metaphase exists. In addition, Ras, like Mos and cytostatic factor, can arrest Xopus embryonic cell cleavage in mitosis and maintain high levels of MPF. Thus, in the Xenpus oocyte and embryo systems Ras functions in the M phase of the cell cycle. The embryonic cleavage arrest assay is a rapid and sensitive test for Ras fiuction.

Proceedings of the National Academy of Sciences of the United States of America, May 15, 1993

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras pr... more Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein. We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with nontransfected cells. Insulin stimulated hexose uptake by-2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras6lK-expressing fibroblasts. Overexpression of N-Ras6lK also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras6lK_ expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras6lK-expressing compared with parental adipocytes. However, expression of N-Ras6lK in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras6lK expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.

Neuroscience, Apr 1, 2007

... ontology; HRP, horseradish peroxidase; IHC, immunohistochemistry/immunohistochemical; LCM, la... more ... ontology; HRP, horseradish peroxidase; IHC, immunohistochemistry/immunohistochemical; LCM, laser capture microdissection; LTD, long term depression; LTP, long term potentiation; NFT, neurofibrillary tangles; RMA, robust microarray analysis; SAM, significance analysis of ...

Journal of Biological Chemistry, Jul 1, 1988