Genomic Profiling of Patient-derived Xenografts for Lung Cancer Identifies b2m Inactivation Impairing Immunorecognition.
Carolina Pereira, Pol Gimenez-Xavier, Eva Pros, Maria J. Pajares, Massimo Moro, Antonio Gomez, Alejandro Navarro, Enric Condom, Sebastian Moran, Gonzalo Gómez-López, Osvaldo Graña, Miriam Rubio-Camarillo, Alex Martinez-Martí, Jun Yokota, Julian Carretero, Jose M Galbis, Ernest Nadal, David G Pisano, Gabriella Sozzi, Enriqueta Felip, Luis M. Montuenga, Luca Roz, Alberto Villanueva and Montse Sanchez-Céspedes
Clin Cancer Res. 2016 Dec 21. pii: clincanres.1946.2016. doi: 10.1158/1078-0432.
We aimed to maximize the performance of detecting genetic alterations in lung cancer (LC) using high-throughput sequencing for patient-derived xenografts (PDXs). EXPERIMENTAL DESIGN:We undertook an integrated RNA and whole-exome sequencing of 14 PDXs. We focused on the genetic and functional analysis of B2-microglobulin (B2M), a component of the HLA class-I complex. RESULTS:We identified alterations in genes involved in various functions, such as B2M involved in immunosurveillance. We extended the mutational analysis of B2M to about 230 LCs. Five per cent of the LCs carried somatic mutations, most of which impaired the correct formation of the HLA-I complex. We also report that genes such as CALR, PDIA3 and TAP1, which are involved in the maturation of the HLA-I complex, are altered in LC. By gene expression microarrays, we observed that restitution of B2M in LC cells up-regulated targets of IFNalpha/IFNgamma. Furthermore, one third of the LCs lacked the HLA-I complex, which was associated with lower cytotoxic CD8+ lymphocyte infiltration. The levels of B2M and HLA-I proteins correlated with those of PD-L1. Finally, a deficiency in HLA-I complex and CD8+ infiltration tended to correlate with reduced survival of LC patients treated with anti-PD1-/anti-PD-L1. CONCLUSIONS:Here, we report recurrent inactivation of B2M in LC. These observations, coupled with the mutations found at CALR, PDIA3 and TAP1, and the down-regulation of the HLA-I complex, indicate that an abnormal immunosurveillance axis contributes to LC development. Finally, our observations suggest that an impaired HLA-I complex affects the response to anti-PD-1/anti-PD-L1 therapies.
Further info:Clinical Cancer Research
HDAC Inhibitors As Novel Targeted Therapies for NUP98-HOXA9 AML Patients.
Ana Rio-Machin, Gonzalo Gómez-López, Alba Maiques-Diaz, Sara Alvarez, Maria Jose Calasanz, Jude Fitzgibbon and Juan C Cigudosa.
Blood 2016 128(22); 2685.
Background: The chromosomal translocation t(7;11)(p15,p15) encodes the oncogenic transcription factor NUP98-HOXA9 which results in a fusion of the nucleoporin 98kDa (NUP98) and homeobox A9 (HOXA9) genes. The oncogenic mechanisms underlying this translocation remain poorly understood and patients are currently inadequately served by traditional cytotoxic chemotherapy regimens.
Aims:To decipher the underlying biology of the NUP98-HOXA9 fusion protein and develop rational therapeutic strategies targeting its oncogenic mechanism. Methods: Human cellular models expressing NUP98-HOXA9, HOXA9 wt or NUP98 wt were established by retroviral transduction of HEK293FT human cell line and human hematopoietic progenitors (CD34+, hHP) isolated from donor cord blood. Chromatin immunoprecepitation experiments followed by sequencing (ChIP-seq) and quantitative ChIP (qChIP) were used to define fusion specific binding locations. Cloning regulatory regions of selected target genes in a luciferase vectorconfirmed the direct involvement of NUP98-HOXA9 in their regulation. RTQ-PCR and gene expression microarrays were used to evaluate expression levels. Co-Immunoprecipitation experiments validated protein-protein interactions and drug treatments were performed at IC50. Cell viability was analysed by apoptosis, proliferation and Colony Forming Unit assays.
Results:Comparison of ChIP-seq data from HEK293FTmodels of NUP98-HOXA9, HOXA9 wt or NUP98 wt respectively, identified 4,471 target genomic regions of the fusion protein (FDR < 0.05), located within +4/-4 kb from the annotated Transcription Start Site (TSS) of 1,363 genes, with 399 genes common to HOXA9 wt and 5 to NUP98 wt. The NUP98-HOXA9 binding sites included enhancers of MEIS1, HOXA9 and PBX3 (PBX3 and HOXA9 were common to NUP98 wt and MEIS1 to HOXA9 wt). Together these transcription factors form a key activator complex that regulates the expression of genes involved in leukemogenesis and its overexpression is significant related to adverse prognosis in AML. Luciferase assays showed that the upregulation of this leukemic axis was directly induced by the interaction of NUP98-HOXA9 with the corresponding enhancer regions of MEIS1, HOXA9 and PBX3. Treatment of cells with HXR9, a specific peptide inhibitor of HOXA9 and PBX3 interaction, led to a selective decrease in the proliferation of hHP expressing NUP98-HOXA9, confirming the relevance of these target genes to its oncogenic mechanism. Combining ChIP-seq and gene expression data of three independent clones of hHP expressing NUP98-HOXA9 and patient samples (n = 5) harbouring t(7;11)(p15,p15) revealed a dual regulatory role of the fusion protein, in both repressing and activating target gene transcription where, for example, MEIS1, HOXA9, PBX3 and AFF3 were found overexpressed and BIRC3, SMAD1, FILIP1L and PTEN downregulated. Interactions of NUP98-HOXA9 with p300 and HDAC1 were shown to drive this transcriptional activation and repression, respectively. We found using qChIP experiments that p300 bound to the regulatory regions of the overexpressed genes only when NUP98-HOXA9 was present, whereas we observed significant enrichment of HDAC1 binding to the promoter regions of the downregulated genes when the fusion protein was expressed. Taking advantage of this latter observation, we demonstrated a dramatic inhibitory effect on the viability of hHP expressing NUP98-HOXA9after the treatment with subtherapeutic doses (IC50 = 4nM) of the HDAC inhibitor LBH-589 (Panobinostat) with no effect in control hHP transduced with an empty vector.
Conclusion: An improved understanding of the pathobiology underlying recurrent translocation events in AML is a critical first step for the development of rational, targeted therapies. Here, we identify upregulation of the targetable MEIS1-HOXA9-PBX3 complex underpinning the leukemogenic activity of NUP98-HOXA9. Its activity in repressing transcription mediated through interaction with HDAC1, has been shown to be also a key pathogenic mechanism that can be exploited through use of HDAC inhibitors and potentially lead to a promising new therapy for this high-risk group of patients.
Further info: Blood
Tissue damage and senescence provide critical signals for cellular reprogramming in vivo.
Mosteiro L, Pantoja C, Alcazar N, Marión RM, Chondronasiou D, Rovira M, Fernandez-Marcos PJ, Muñoz-Martin M, Blanco-Aparicio C, Pastor J, Gómez-López G, De Martino A, Blasco MA, Abad M, Serrano M.
Science 2016 Nov 25;354(6315)
Reprogramming of differentiated cells into pluripotent cells can occur in vivo, but the mechanisms involved remain to be elucidated. Senescence is a cellular response to damage, characterized by abundant production of cytokines and other secreted factors that, together with the recruitment of inflammatory cells, result in tissue remodeling. Here, we show that in vivo expression of the reprogramming factors OCT4, SOX2, KLF4, and cMYC (OSKM) in mice leads to senescence and reprogramming, both coexisting in close proximity. Genetic and pharmacological analyses indicate that OSKM-induced senescence requires the Ink4a/Arf locus and, through the production of the cytokine interleukin-6, creates a permissive tissue environment for in vivo reprogramming. Biological conditions linked to senescence, such as tissue injury or aging, favor in vivo reprogramming by OSKM. These observations may be relevant for tissue repair.
Further info: Science
RUbioSeq+: A multiplatform application that executes parallelized pipelines to analyse next-generation sequencing data.
Rubio-Camarillo M, López-Fernández H, Gómez-López G, Carro A, Fernández JM, Torre CF, Fdez-Riverola F, Glez-Peña D.
Comput Methods Programs Biomed. 2017 Jan;138:73-81. doi: 10.1016/j.cmpb.2016.10.008.
BACKGROUND AND OBJECTIVE:To facilitate routine analysis and to improve the reproducibility of the results, next-generation sequencing (NGS) analysis requires intuitive, efficient and integrated data processing pipelines. METHODS:We have selected well-established software to construct a suite of automated and parallelized workflows to analyse NGS data for DNA-seq (single-nucleotide variants (SNVs) and indels), CNA-seq, bisulfite-seq and ChIP-seq experiments. RESULTS:Here, we present RUbioSeq+, an updated and extended version of RUbioSeq, a multiplatform application that incorporates a suite of automated and parallelized workflows to analyse NGS data. This new version includes: (i) an interactive graphical user interface (GUI) that facilitates its use by both biomedical researchers and bioinformaticians, (ii) a new pipeline for ChIP-seq experiments, (iii) pair-wise comparisons (case-control analyses) for DNA-seq experiments, (iv) and improvements in the parallelized and multithreaded execution options. Results generated by our software have been experimentally validated and accepted for publication. CONCLUSIONS:RUbioSeq+ is free and open to all users at http://rubioseq.bioinfo.cnio.es/.
Further info: Computer Methods and Programs in Biomedicine
Lineage-specific roles of the cytoplasmic polyadenylation factor CPEB4 in the regulation of melanoma drivers.
Pérez-Guijarro E, Karras P, Cifdaloz M, Martínez-Herranz R, Cañón E, Graña O, Horcajada-Reales C, Alonso-Curbelo D, Calvo TG, Gómez-López G, Bellora N, Riveiro-Falkenbach E, Ortiz-Romero PL, Rodríguez-Peralto JL, Maestre L, Roncador G, de Agustín Asensio JC, Goding CR, Eyras E, Megías D, Méndez R, Soengas MS.
Nat Commun. 2016 Nov 18;7:13418. doi: 10.1038/ncomms13418.
Nuclear 3'-end-polyadenylation is essential for the transport, stability and translation of virtually all eukaryotic mRNAs. Poly(A) tail extension can also occur in the cytoplasm, but the transcripts involved are incompletely understood, particularly in cancer. Here we identify a lineage-specific requirement of the cytoplasmic polyadenylation binding protein 4 (CPEB4) in malignant melanoma. CPEB4 is upregulated early in melanoma progression, as defined by computational and histological analyses. Melanoma cells are distinct from other tumour cell types in their dependency on CPEB4, not only to prevent mitotic aberrations, but to progress through G1/S cell cycle checkpoints. RNA immunoprecipitation, sequencing of bound transcripts and poly(A) length tests link the melanoma-specific functions of CPEB4 to signalling hubs specifically enriched in this disease. Essential in these CPEB4-controlled networks are the melanoma drivers MITF and RAB7A, a feature validated in clinical biopsies. These results provide new mechanistic links between cytoplasmic polyadenylation and lineage specification in melanoma.
Further info: Nature Communications
Pax8 controls thyroid follicular polarity through Cadherin-16.
Koumarianou P, Gonzalo Gómez-López, Santisteban P.
J Cell Sci.2016 Oct 25. pii: jcs.184291.
Organization of epithelial cells during follicular lumen formation is crucial for thyroid morphogenesis and function of the thyroid gland; however, the molecular mechanisms underlying this are poorly understood. To investigate this process, we established three-dimensional (3D) epithelial culture model systems using Fischer rat thyroid (FRT) cells or murine primary thyrocytes that developed polarized spherical structures with a central lumen mimicking thyroid follicles. Using microarray-based differential expression analysis of FRT cells grown under 2D or 3D conditions followed by RNA-mediated interference (RNAi) and morphogenetic analysis, we identified a key role for the thyroid transcription factor Pax8 and its target Cadherin-16 (Cdh16) in the generation of polarized follicle-like structures. Silencing Pax8 expression inhibited the acquisition of apical-basal membrane polarity and impaired lumen formation. Both laminin and β1-integrin (Itgb1) expression was reduced, while cell cytoskeleton polarized distribution was altered. Silencing Cdh16 expression also led to the formation of defective structures characterized by very low laminin expression at the follicle-matrix interface, down-regulation of Itgb1, and unpolarized distribution of cell cytoskeleton. Our results demonstrate that Pax8 controls apical-basal follicular polarization and follicle formation through Cdh16.
Further info: Journal of Cell Science
p21Cip1 plays a critical role in the physiological adaptation to fasting through activation of PPARalpha.
Elena Lopez-Guadamillas, Pablo J. Fernandez-Marcos, Cristina Pantoja, Maribel Muñoz-Martin, Dolores Martínez, Gonzalo Gómez-López, Ramón Campos-Olivas, Angela M. Valverde, Manuel Serrano.
Scientific Reports 6, Article number: 34542 (2016).
Fasting is a physiological stress that elicits well-known metabolic adaptations, however, little is known about the role of stress-responsive tumor suppressors in fasting. Here, we have examined the expression of several tumor suppressors upon fasting in mice. Interestingly, p21 mRNA is uniquely induced in all the tissues tested, particularly in liver and muscle (>10 fold), and this upregulation is independent of p53. Remarkably, in contrast to wild-type mice, p21-null mice become severely morbid after prolonged fasting. The defective adaptation to fasting of p21-null mice is associated to elevated energy expenditure, accelerated depletion of fat stores, and premature activation of protein catabolism in the muscle. Analysis of the liver transcriptome and cell-based assays revealed that the absence of p21 partially impairs the transcriptional program of PPARα, a key regulator of fasting metabolism. Finally, treatment of p21-null mice with a PPARα agonist substantially protects them from their accelerated loss of fat upon fasting. We conclude that p21 plays a relevant role in fasting adaptation through the positive regulation of PPARα.
Further info:Scientific Reports
KRAS-driven lung adenocarcinoma: combined DDR1/Notch inhibition as an effective therapy
Chiara Ambrogio, Ernest Nadal, Alberto Villanueva, Gómez-López G, Timothy P Cash, Mariano Barbacid, David Santamaría
ESMO Open 2016 doi:10.1136/esmoopen-2016-000076
Understanding the early evolution of cancer heterogeneity during the initial steps of tumorigenesis can uncover vulnerabilities of cancer cells that may be masked at later stages. We describe a comprehensive approach employing gene expression analysis in early lesions to identify novel therapeutic targets and the use of mouse models to test synthetic lethal drug combinations to treat human Kirsten rat sarcoma viral oncogene homologue (KRAS)-driven lung adenocarcinoma.
Further info: ESMO Open
NSD2 contributes to oncogenic RAS-driven transcription in lung cancer cells through long-range epigenetic activation.
Verónica García-Carpizo, Jacinto Sarmentero, Bomie Han, Osvaldo Graña, Sergio Ruiz-Llorente, David G. Pisano, Manuel Serrano, Harold B. Brooks, Robert M. Campbell, Maria J. Barrero.
Scientific Reports 2016; 6, 32952. doi: 10.1038/srep32952
The histone methyltransferase NSD2/WHSC1/MMSET is overexpressed in a number of solid tumors but its contribution to the biology of these tumors is not well understood. Here, we describe that NSD2 contributes to the proliferation of a subset of lung cancer cell lines by supporting oncogenic RAS transcriptional responses. NSD2 knock down combined with MEK or BRD4 inhibitors causes co-operative inhibitory responses on cell growth. However, while MEK and BRD4 inhibitors converge in the downregulation of genes associated with cancer-acquired super-enhancers, NSD2 inhibition affects the expression of clusters of genes embedded in megabase-scale regions marked with H3K36me2 and that contribute to the RAS transcription program. Thus, combinatorial therapies using MEK or BRD4 inhibitors together with NSD2 inhibition are likely to be needed to ensure a more comprehensive inhibition of oncogenic RAS-driven transcription programs in lung cancers with NSD2 overexpression.
Further info: Scientific Reports
A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance.
Martínez P, Gómez-López G, Pisano DG, Flores JM, Blasco MA.
Aging Cell 2016 Sep 1. doi: 10.1111/acel.12517.
RAP1 is one of the components of shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere length maintenance and protection has remained elusive. RAP1 also binds subtelomeric repeats and along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres, and its deficiency causes a premature aging in humans and mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact on mouse survival, mice lacking both RAP1 and telomerase show a progressively decreased survival with increasing mouse generations compared to telomerase single mutants. Telomere shortening is more pronounced in Rap1-/- Terc-/- doubly deficient mice than in the single-mutant Terc-/- counterparts, leading to an earlier onset of telomere-induced DNA damage and degenerative pathologies. Telomerase deficiency abolishes obesity and liver steatohepatitis provoked by RAP1 deficiency. Using genomewide ChIP sequencing, we find that progressive telomere shortening owing to telomerase deficiency leads to re-localization of RAP1 from telomeres and subtelomeric regions to extratelomeric sites in a genomewide manner. These findings suggest that although in the presence of sufficient telomere reserve RAP1 is not a key factor for telomere maintenance and protection, it plays a crucial role in the context of telomerase deficiency, thus in agreement with its evolutionary conservation as a telomere component from yeast to humans.
Further info: Aging Cell
Telomeric RNAs are essential to maintain telomeres.
Montero JJ, López de Silanes I, Graña O, Blasco MA.
Nat Commun 2016 Aug 17;7:12534. doi: 10.1038/ncomms12534.
Telomeres are transcribed generating long non-coding RNAs known as TERRA. Deciphering the role of TERRA has been one of the unsolved issues of telomere biology in the past decade. This has been, in part, due to lack of knowledge on the TERRA loci, thus preventing functional genetic studies. Here, we describe that long non-coding RNAs with TERRA features are transcribed from the human 20q and Xp subtelomeres. Deletion of the 20q locus by using the CRISPR-Cas9 technology causes a dramatic decrease in TERRA levels, while deletion of the Xp locus does not result in decreased TERRA levels. Strikingly, 20q-TERRA ablation leads to dramatic loss of telomere sequences and the induction of a massive DNA damage response. These findings identify chromosome 20q as a main TERRA locus in human cells and represent the first demonstration in any organism of the essential role of TERRA in the maintenance of telomeres.
Further info: Nature Communications
Metastatic risk and resistance to BRAF inhibitors in melanoma defined by selective allelic loss of ATG5.
García-Fernández M, Karras P, Checinska A, Cañón E, Calvo GT, Gómez-López G, Cifdaloz M, Colmenar A, Espinosa-Hevia L, Olmeda D, Soengas MS.
Autophagy 2016 Jul 27:1-15 doi: 10.1080/15548627.2016.1199301
Melanoma is a paradigm of aggressive tumors with a complex and heterogeneous genetic background. Still, melanoma cells frequently retain developmental traits that trace back to lineage specification programs. In particular, lysosome-associated vesicular trafficking is emerging as a melanoma-enriched lineage dependency. However, the contribution of other lysosomal functions such as autophagy to melanoma progression is unclear, particularly in the context of metastasis and resistance to targeted therapy. Here we mined a broad spectrum of cancers for a meta-analysis of mRNA expression, copy number variation and prognostic value of 13 core autophagy genes. This strategy identified heterozygous loss of ATG5 at chromosome band 6q21 as a distinctive feature of advanced melanomas. Importantly, partial ATG5 loss predicted poor overall patient survival in a manner not shared by other autophagy factors and not recapitulated in other tumor types. This prognostic relevance of ATG5 copy number was not evident for other 6q21 neighboring genes. Melanocyte-specific mouse models confirmed that heterozygous (but not homozygous) deletion of Atg5 enhanced melanoma metastasis and compromised the response to targeted therapy (exemplified by dabrafenib, a BRAF inhibitor in clinical use). Collectively, our results support ATG5 as a therapeutically relevant dose-dependent rheostat of melanoma progression. Moreover, these data have important translational implications in drug design, as partial blockade of autophagy genes may worsen (instead of counteracting) the malignant behavior of metastatic melanomas.
Further info: Autophagy
Deep sequencing reveals microRNAs predictive of antiangiogenic drug response.
Jesús García-Donas, Benoit Beuselinck, Lucía Inglada-Pérez, Osvaldo Graña, Patrick Schöffski, Agnieszka Wozniak, Oliver Bechter, Maria Apellániz-Ruiz, Luis Javier Leandro-García, Emilio Esteban, Daniel E. Castellano, Aranzazu González del Alba, Miguel Angel Climent, Susana Hernando, José Angel Arranz, Manuel Morente, David G Pisano Mercedes Robledo and Cristina Rodriguez-Antona.
JCI Insight 2016;1(10):e86051. doi:10.1172/jci.insight.86051.
The majority of metastatic renal cell carcinoma (RCC) patients are treated with tyrosine kinase inhibitors (TKI) in first-line treatment; however, a fraction are refractory to these antiangiogenic drugs. MicroRNAs (miRNAs) are regulatory molecules proven to be accurate biomarkers in cancer. Here, we identified miRNAs predictive of progressive disease under TKI treatment through deep sequencing of 74 metastatic clear cell RCC cases uniformly treated with these drugs. Twenty-nine miRNAs were differentially expressed in the tumors of patients who progressed under TKI therapy (P values from 6 × 10–9 to 3 × 10–3). Among 6 miRNAs selected for validation in an independent series, the most relevant associations corresponded to miR–1307-3p, miR–155-5p, and miR–221-3p (P = 4.6 × 10–3, 6.5 × 10–3, and 3.4 × 10–2, respectively). Furthermore, a 2 miRNA–based classifier discriminated individuals with progressive disease upon TKI treatment (AUC = 0.75, 95% CI, 0.64–0.85; P = 1.3 × 10–4) with better predictive value than clinicopathological risk factors commonly used. We also identified miRNAs significantly associated with progression-free survival and overall survival (P = 6.8 × 10–8 and 7.8 × 10–7 for top hits, respectively), and 7 overlapped with early progressive disease. In conclusion, this is the first miRNome comprehensive study, to our knowledge, that demonstrates a predictive value of miRNAs for TKI response and provides a new set of relevant markers that can help rationalize metastatic RCC treatment.
Further info: JCI Insight
Proteomic Profiling of Ewing Sarcoma Reveals a Role for TRAF6 in Proliferation and Ribonucleoproteins/RNA Processing.
Madoz-Gúrpide J, Herrero-Martín D, Gómez-López G, Hontecillas-Prieto L, Biscuola M, Chamizo C, García-Domínguez D, Marcilla D, Amaral AT, Ordóñez JL and De Álava E.
J Proteomics Bioinform (2016) 9:166-175. doi:10.4172/jpb.1000403
Notwithstanding advances over the last decade in the comprehension of the molecular biology of Ewing sarcoma (ES), we still lack an understanding of critical issues, especially those regarding its genesis. In particular, little effort has been devoted to characterization of the proteic component of the mechanisms and pathways deployed by the activation of the fusion protein resulting from chromosomal translocation. We decided to investigate the proteic alterations of an ES cell line bearing a representative fusion protein. The combination of RNA interference of EWSFLI1 in the ES cell line TC-71, a proteomic analysis by 2-D electrophoresis and subsequent mass-spectrometry identification, and a global overrepresentation study detected changes in more than 500 spots. Forty-three proteins were identified as being significantly differentially abundant. As expected, we found and validated changes in proteins linked to nucleotide processing, transcription regulation, ribonucleoproteins, helicases, cell-cycle control and proliferation, and metabolic processes. Additionally, TNF receptor-associated factor 6 was revealed as a hub node. Our strategy showed the potential to reveal the protein interplay associated with the known functions of the fusion protein: binding to DNA and RNA in order to act as aberrant transcription factors or potent repressors, or by altering RNA processing.
Further info: J Proteomics Bioinform
Targeting Tumor Mitochondrial Metabolism Overcomes Resistance to Antiangiogenics.
Paloma Navarro, Maria J. Bueno, Ivana Zagorac, Tamara Mondejar, Jesus Sanchez, Silvana Mourón, Javier Muñoz, Gonzalo Gómez-López, Veronica Jimenez-Renard, Francisca Mulero, Navdeep S. Chandel, Miguel Quintela-Fandino.
Cell Reports 2016 doi:10.1016/j.celrep.2016.05.052
Epithelial malignancies are effectively treated by antiangiogenics; however, acquired resistance is a major problem in cancer therapeutics. Epithelial tumors commonly have mutations in the MAPK/Pi3K-AKT pathways, which leads to high-rate aerobic glycolysis. Here, we show how multikinase inhibitor antiangiogenics (TKIs) induce hypoxia correction in spontaneous breast and lung tumor models. When this happens, the tumors downregulate glycolysis and switch to long-term reliance on mitochondrial respiration. A transcriptomic, metabolomic, and phosphoproteomic study revealed that this metabolic switch is mediated by downregulation of HIF1α and AKT and upregulation of AMPK, allowing uptake and degradation of fatty acids and ketone bodies. The switch renders mitochondrial respiration necessary for tumor survival. Agents like phenformin or ME344 induce synergistic tumor control when combined with TKIs, leading to metabolic synthetic lethality. Our study uncovers mechanistic insights in the process of tumor resistance to TKIs and may have clinical applicability.
Further info: Cell Reports
Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance.
Manso L, Mourón S, Tress M, Gómez-López G, Morente M, Ciruelos E, Rubio-Camarillo M, Rodriguez-Peralto JL, Pujana MA, Pisano DG, Quintela-Fandino M.
PLoS One. May 19;11(5):e0155840.
We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25, and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.
Further info: PLoS One
Vitamin D receptor expression and associated gene signature in tumour stromal fibroblasts predict clinical outcome in colorectal cancer
Gemma Ferrer-Mayorga, Gonzalo Gómez-López, Antonio Barbáchano,Asunción Fernández-Barral, Cristina Peña, David G Pisano, Ramón Cantero, Federico Rojo, Alberto Muñoz, María Jesús Larriba.
Gut 2016 April 6, 2016 as 10.1136/gutjnl-2015-310977
Objective: Colorectal cancer (CRC) is a major health concern. Vitamin D deficiency is associated with high CRC incidence and mortality, suggesting a protective effect of vitamin D against this disease. Given the strong influence of tumour stroma on cancer progression, we investigated the potential effects of the active vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) on CRC stroma. Design: Expression of vitamin D receptor (VDR) and two 1,25(OH)2D3 target genes was analysed in 658 patients with CRC with prolonged clinical followup. 1,25(OH)2D3 effects on primary cultures of patientderived colon normal fibroblasts (NFs) and cancerassociated fibroblasts (CAFs) were studied using collagen gel contraction and migration assays and global gene expression analyses. Publicly available data sets (n=877) were used to correlate the 1,25(OH)2D3-associated gene signature in CAFs with CRC outcome. Results: High VDR expression in tumour stromal fibroblasts was associated with better overall survival(OS) and progression-free survival in CRC, independently of its expression in carcinoma cells. 1,25(OH)2D3 inhibited the protumoural activation of NFs and CAFs and imposed in CAFs a 1,25(OH)2D3-associated gene signature that correlated with longer OS and disease-free survival in CRC. Furthermore, expression of two genes from the signature, CD82 and S100A4, correlated with stromal VDR expression and clinical outcome in our cohort of patients with CRC. Conclusions: 1,25(OH)2D3 has protective effects against CRC through the regulation of stromal fibroblasts. Accordingly, expression of VDR and 1,25 (OH)2D3-associated gene signature in stromal fibroblasts predicts a favourable clinical outcome in CRC. Therefore, treatment of patients with CRC with VDR agonists could be explored even in the absence of VDR expression in carcinoma cells.
Further info: Gut
Chronic skin inflammation leads to bone loss by IL-17–mediated inhibition of Wnt signaling in osteoblasts.
Özge Uluçkan, Maria Jimenez, Susanne Karbach, Anke Jeschke, Osvaldo Graña, Johannes Keller, Björn Busse, Andrew L. Croxford, Stephanie Finzel, Marije Koenders, Wim van den Berg, Thorsten Schinke, Michael Amling, Ari Waisman, Georg Schett, Erwin F. Wagner.
Science Translational Medicine 16 Mar 2016: Vol. 8, Issue 330, pp. 330ra37,DOI: 10.1126/scitranslmed.aad8996
Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A–mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A–mediated skin inflammation, K14-IL17Aind and JunBΔep, strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients.
Further info: Science Translational Medicine
Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma.
Ambrogio C, Gómez-López G, Falcone M, Vidal A, Nadal E, Crosetto N, Blasco RB, Fernández-Marcos PJ, Sánchez-Céspedes M, Ren X, Wang Z, Ding K, Hidalgo M, Serrano M, Villanueva A, Santamaría D, Barbacid M.
Nature Medicine 2016 Feb 8. doi: 10.1038/nm.4041.
Patients with advanced Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant lung adenocarcinoma are currently treated with standard chemotherapy because of a lack of efficacious targeted therapies. We reasoned that the identification of mediators of Kras signaling in early mouse lung hyperplasias might bypass the difficulties that are imposed by intratumor heterogeneity in advanced tumors, and that it might unveil relevant therapeutic targets. Transcriptional profiling of KrasG12V-driven mouse hyperplasias revealed intertumor diversity with a subset that exhibited an aggressive transcriptional profile analogous to that of advanced human adenocarcinomas. The top-scoring gene in this profile encodes the tyrosine kinase receptor DDR1. The genetic and pharmacological inhibition of DDR1 blocked tumor initiation and tumor progression, respectively. The concomitant inhibition of both DDR1 and Notch signaling induced the regression of KRAS;TP53-mutant patient-derived lung xenografts (PDX) with a therapeutic efficacy that was at least comparable to that of standard chemotherapy. Our data indicate that the combined inhibition of DDR1 and Notch signaling could be an effective targeted therapy for patients with KRAS-mutant lung adenocarcinoma.
Further info: Nature Medicine
Partial Loss of Rpl11 in Adult Mice Recapitulates Diamond-Blackfan Anemia and Promotes Lymphomagenesis.
Lucia Morgado-Palacin, Gianluca Varetti, Susana Llanos, Gonzalo Gómez-López, Dolores Martinez, Manuel Serrano.
Cell Reports 13, 1–11, October 27, 2015. doi:10.1016/j.celrep.2015.09.038
Diamond-Blackfan anemia (DBA) is characterized by anemia and cancer susceptibility and is caused by mutations in ribosomal genes, including RPL11. Here, we report that Rpl11-heterozygous mouse embryos are not viable and that Rpl11 homozygous deletion in adult mice results in death within a few weeks, accompanied by bone marrow aplasia and intestinal atrophy. Importantly, Rpl11 heterozygous deletion in adult mice results in anemia associated with decreased erythroid progenitors and defective erythroid maturation. These defects are also present in mice transplanted with inducible heterozygous Rpl11 bone marrow and, therefore, are intrinsic to the hematopoietic system. Additionally, heterozygous Rpl11 mice present increased susceptibility to radiation-induced lymphomagenesis. In this regard, total or partial deletion of Rpl11 compromises p53 activation upon ribosomal stress or DNA damage in fibroblasts. Moreover, fibroblasts and hematopoietic tissues from heterozygous Rpl11 mice present higher basal cMYC levels. We conclude that Rpl11-deficient mice recapitulate DBA disorder, including cancer predisposition.
Further info: Cell Reports
MYC/PGC-1α Balance Determines the Metabolic Phenotype and Plasticity of Pancreatic Cancer Stem Cells.
Sancho P, Burgos-Ramos E, Tavera A, Bou Kheir T, Jagust P, Schoenhals M, Barneda D, Sellers K, Campos-Olivas R, Graña O, Viera CR, Yuneva M, Sainz B Jr, Heeschen C.
Cell Metabolism 2015 Sep 9. pii: S1550-4131(15)00406-4.
The anti-diabetic drug metformin targets pancreatic cancer stem cells (CSCs), but not their differentiated progenies (non-CSCs), which may be related to distinct metabolic phenotypes. Here we conclusively demonstrate that while non-CSCs were highly glycolytic, CSCs were dependent on oxidative metabolism (OXPHOS) with very limited metabolic plasticity. Thus, mitochondrial inhibition, e.g., by metformin, translated into energy crisis and apoptosis. However, resistant CSC clones eventually emerged during treatment with metformin due to their intermediate glycolytic/respiratory phenotype. Mechanistically, suppression of MYC and subsequent increase of PGC-1α were identified as key determinants for the OXPHOS dependency of CSCs, which was abolished in resistant CSC clones. Intriguingly, no resistance was observed for the mitochondrial ROS inducer menadione and resistance could also be prevented/reversed for metformin by genetic/pharmacological inhibition of MYC. Thus, the specific metabolic features of pancreatic CSCs are amendable to therapeutic intervention and could provide the basis for developing more effective therapies to combat this lethal cancer.
Further info: Cell Metabolism
Profiling of Sox4-dependent transcriptome in skin links tumour suppression and adult stem cell activation.
Miguel Foronda, Lucía Morgado-Palacin, Gonzalo Gómez-López, Orlando Domínguez, David G. Pisano, María A. Blasco.
Genomics Data 2015, Vol. 6, Pages 21-24.
Adult stem cells (ASCs) reside in specific niches in a quiescent state in adult mammals. Upon specific cues they become activated and respond by self-renewing and differentiating into newly generated specialised cells that ensure appropriate tissue fitness. ASC quiescence also serves as a tumour suppression mechanism by hampering cellular transformation and expansion (White AC et al., 2014). Some genes restricted to early embryonic development and adult stem cell niches are often potent modulators of stem cell quiescence, and derailed expression of these is commonly associated to cancer (Vervoort SJ et al., 2013). Among them, it has been shown that recommissioned Sox4 expression facilitates proliferation, survival and migration of malignant cells. By generating a conditional Knockout mouse model in stratified epithelia (Sox4cKO mice), we demonstrated a delayed plucking-induced Anagen in the absence of Sox4. Skin global transcriptome analysis revealed a prominent defect in the induction of transcriptional networks that control hair follicle stem cell (HFSC) activation such as those regulated by Wnt/Ctnnb1, Shh, Myc or Sox9, cell cycle and DNA damage response-associated pathways. Besides, Sox4cKO mice are resistant to skin carcinogenesis, thus linking Sox4 to both normal and pathological HFSC activation (Foronda M et al., 2014). Here we provide additional details on the analysis of Sox4-regulated transcriptome in Telogen and Anagen skin. The raw and processed microarray data is deposited in GEO under GSE58155.
Further info: Genomics Data
Non-coding recurrent mutations in chronic lymphocytic leukaemia.
Puente XS, Beà S, Valdés-Mas R, Villamor N, Gutiérrez-Abril J, Martín-Subero JI, Munar M, Rubio-Pérez C, Jares P, Aymerich M, Baumann T, Beekman R, Belver L, Carrio A, Castellano G, Clot G, Colado E, Colomer D, Costa D, Delgado J, Enjuanes A, Estivill X, Ferrando AA, Gelpí JL, González B, González S, González M, Gut M, Hernández-Rivas JM, López-Guerra M, Martín-García D, Navarro A, Nicolás P, Orozco M, Payer ÁR, Pinyol M, Pisano DG, Puente DA, Queirós AC, Quesada V, Romeo-Casabona CM, Royo C, Royo R, Rozman M, Russiñol N, Salaverría I, Stamatopoulos K, Stunnenberg HG, Tamborero D, Terol MJ, Valencia A, López-Bigas N, Torrents D, Gut I, López-Guillermo A, López-Otín C, Campo E.
Nature 2015 Jul 22. doi: 10.1038/nature14666.
Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.
Further info: Nature
iMSRC: converting a standard automated microscope into an intelligent screening platform.
Carro A, Perez-Martínez M, Soriano J, Pisano DG, Megías D.
Sci Rep 2015 May 27;5:10502. doi: 10.1038/srep10502.
Microscopy in the context of biomedical research is demanding new tools to automatically detect and capture objects of interest. The few extant packages addressing this need, however, have enjoyed limited uptake due to complexity of use and installation. To overcome these drawbacks, we developed iMSRC, which combines ease of use and installation with high flexibility and enables applications such as rare event detection and high-resolution tissue sample screening, saving time and resources.
Further info: Scientific Reports
DNA repair capacity is impaired in healthy BRCA1 heterozygous mutation carriers.
Vaclová T, Gómez-López G, YSetién F, Bueno JM, Macías JA, Barroso A, Urioste M, Esteller M, Benítez J, Osorio A.
Breast Cancer Res Treat. 2015 Jun 14.
BRCA1 germline mutations increase the lifetime risk of developing breast and ovarian cancers. However, taking into account the differences in disease manifestation among mutation carriers, it is probable that different BRCA1 mutations have distinct haploinsufficiency effects and lead to the formation of different phenotypes. Using lymphoblastoid cell lines derived from heterozygous BRCA1 mutation carriers and non-carriers, we investigated the haploinsufficiency effects of various mutation types using qPCR, immunofluorescence, and microarray technology. Lymphoblastoid cell lines carrying a truncating mutation showed significantly lower BRCA1 mRNA and protein levels and higher levels of gamma-H2AX than control cells or those harboring a missense mutation, indicating greater spontaneous DNA damage. Cells carrying either BRCA1 mutation type showed impaired RAD51 foci formation, suggesting defective repair in mutated cells. Moreover, compared to controls, cell lines carrying missense mutations displayed a more distinct expression profile than cells with truncating mutations, which is consistent with different mutations giving rise to distinct phenotypes. Alterations in the immune response pathway in cells harboring missense mutations point to possible mechanisms of breast cancer initiation in carriers of these mutations. Our findings offer insight into how various heterozygous mutations in BRCA1 could lead to impairment of BRCA1 function and provide strong evidence of haploinsufficiency in BRCA1 mutation carriers.
Further info: PubMed
Tumor MicroRNA Expression Profiling Identifies Circulating MicroRNAs for Early Breast Cancer Detection.
Matamala N, Vargas MT, González-Cámpora R, Miñambres R, Arias JI, Menéndez P, Andrés-León E, Gómez-López G, Yanowsky K, Calvete-Candenas J, Inglada-Pérez L, Martínez-Delgado B, Benítez J.
Clin Chem. 2015 Jun 8. pii: clinchem.2015.238691.
The identification of novel biomarkers for early breast cancer detection would be a great advance. Because of their role in tumorigenesis and stability in body fluids, microRNAs (miRNAs) are emerging as a promising diagnostic tool. Our aim was to identify miRNAs deregulated in breast tumors and evaluate the potential of circulating miRNAs in breast cancer detection. METHODS:We conducted miRNA expression profiling of 1919 human miRNAs in paraffin-embedded tissue from 122 breast tumors and 11 healthy breast tissue samples. Differential expression analysis was performed, and a microarray classifier was generated. The most relevant miRNAs were analyzed in plasma from 26 healthy individuals and 83 patients with breast cancer (36 before and 47 after treatment) and validated in 116 healthy individuals and 114 patients before treatment. RESULTS:We identified a large number of miRNAs deregulated in breast cancer and generated a 25-miRNA microarray classifier that discriminated breast tumors with high diagnostic sensitivity and specificity. Ten miRNAs were selected for further investigation, of which 4 (miR-505-5p, miR-125b-5p, miR-21-5p, and miR-96-5p) were significantly overexpressed in pretreated patients with breast cancer compared with healthy individuals in 2 different series of plasma. MiR-505-5p and miR-96-5p were the most valuable biomarkers (area under the curve 0.72). Moreover, the expression levels of miR-3656, miR-505-5p, and miR-21-5p were decreased in a group of treated patients. CONCLUSIONS:Circulating miRNAs reflect the presence of breast tumors. The identification of deregulated miRNAs in plasma of patients with breast cancer supports the use of circulating miRNAs as a method for early breast cancer detection.
Further info: Clinical Chemistry
The pluripotency factor NANOG promotes the formation of squamous cell carcinomas.
Palla AR, Piazzolla D, Alcazar N, Cañamero M, Graña O, Gómez-López G, Dominguez O, Dueñas M, Paramio JM, Serrano M.
Sci Rep. 2015 May 19;5:10205. doi: 10.1038/srep10205.
NANOG is a key pluripotency factor in embryonic stem cells that is frequently expressed in squamous cell carcinomas (SCCs). However, a direct link between NANOG and SCCs remains to be established. Here, we show that inducible overexpression of NANOG in mouse skin epithelia favours the malignant conversion of skin papillomas induced by chemical carcinogenesis, leading to increased SCC formation. Gene expression analyses in pre-malignant skin indicate that NANOG induces genes associated to epithelial-mesenchymal transition (EMT). Some of these genes are directly activated by NANOG, including EMT-associated genes Zeb1, Zeb2, Twist1, Prrx1 and miR-21. Finally, endogenous NANOG binds to the promoters of theses genes in human SCC cells and, moreover, NANOG induces EMT features in primary keratinocytes. These results provide in vivo evidence for the oncogenic role of NANOG in squamous cell carcinomas.
Further info: Scientific Reports
The UBC-40 Urothelial Bladder Cancer cell line index: a genomic resource for functional studies.
Earl J, Rico D, Carrillo-de-Santa-Pau E, Rodríguez-Santiago B, Méndez-Pertuz M, Auer H, Gomez G, Grossman HB, Pisano DG Schulz WA, Pérez-Jurado LA, Carrato A, Theodorescu D, Chanock S, Valencia A, Real FX.
BMC Genomics. 2015 May 22;16(1):403.
BACKGROUND: Urothelial bladder cancer is a highly heterogeneous disease. Cancer cell lines are useful tools for its study. This is a comprehensive genomic characterization of 40 urothelial bladder carcinoma (UBC) cell lines including information on origin, mutation status of genes implicated in bladder cancer (FGFR3, PIK3CA, TP53, and RAS), copy number alterations assessed using high density SNP arrays, uniparental disomy (UPD) events, and gene expression. RESULTS: Based on gene mutation patterns and genomic changes we identify lines representative of the FGFR3-driven tumor pathway and of the TP53/RB tumor suppressor-driven pathway. High-density array copy number analysis identified significant focal gains (1q32, 5p13.1-12, 7q11, and 7q33) and losses (i.e. 6p22.1) in regions altered in tumors but not previously described as affected in bladder cell lines. We also identify new evidence for frequent regions of UPD, often coinciding with regions reported to be lost in tumors. Previously undescribed chromosome X losses found in UBC lines also point to potential tumor suppressor genes. Cell lines representative of the FGFR3-driven pathway showed a lower number of UPD events. CONCLUSIONS: Overall, there is a predominance of more aggressive tumor subtypes among the cell lines. We provide a cell line classification that establishes their relatedness to the major molecularly-defined bladder tumor subtypes. The compiled information should serve as a useful reference to the bladder cancer research community and should help to select cell lines appropriate for the functional analysis of bladder cancer genes, for example those being identified through massive parallel sequencing.
Further info: BMC Genomics
miRGate: a curated database of human, mouse and rat miRNA-mRNA targets.
Andrés-León E, González Peña D, Gomez-Lopez G and Pisano DG.
Database (Oxford). 2015 Apr 8;2015. pii: bav035. doi: 10.1093/database/bav035.
MicroRNAs (miRNAs) are small non-coding elements involved in the post-transcriptional down-regulation of gene expression through base pairing with messenger RNAs (mRNAs). Through this mechanism, several miRNA-mRNA pairs have been described as critical in the regulation of multiple cellular processes, including early embryonic development and pathological conditions. Many of these pairs (such as miR-15 b/BCL2 in apoptosis or BART-6/BCL6 in diffuse large B-cell lymphomas) were experimentally discovered and/or computationally predicted. Available tools for target prediction are usually based on sequence matching, thermodynamics and conservation, among other approaches. Nevertheless, the main issue on miRNA-mRNA pair prediction is the little overlapping results among different prediction methods, or even with experimentally validated pairs lists, despite the fact that all rely on similar principles. To circumvent this problem, we have developed miRGate, a database containing novel computational predicted miRNA-mRNA pairs that are calculated using well-established algorithms. In addition, it includes an updated and complete dataset of sequences for both miRNA and mRNAs 3'-Untranslated region from human (including human viruses), mouse and rat, as well as experimentally validated data from four well-known databases. The underlying methodology of miRGate has been successfully applied to independent datasets providing predictions that were convincingly validated by functional assays. miRGate is an open resource available at http://mirgate.bioinfo.cnio.es. For programmatic access, we have provided a representational state transfer web service application programming interface that allows accessing the database at http://mirgate.bioinfo.cnio.es/API/ Database URL: http://mirgate.bioinfo.cnio.es
Further info: Oxford Database
Nuclear DICKKOPF-1 as a biomarker of chemoresistance and poor clinical outcome in colorectal cancer.
Aguilera Ó, González-Sancho JM, Zazo S, Rincón R, Fernández AF, Tapia O, Canals F, Morte B, Calvanese V, Orgaz JL, Niell N, Aguilar S, Freije JM, Graña O, Pisano DG, Borrero A, Martínez-Useros J, Jiménez B, Fraga MF, García-Foncillas J, López-Otín C, Lafarga M, Rojo F, Muñoz A.
Oncotarget 2015 Mar 20;6(8):5903-17.
Sporadic colorectal cancer (CRC) insurgence and progression depend on the activation of Wnt/β-catenin signaling. Dickkopf (DKK)-1 is an extracellular inhibitor of Wnt/β-catenin signaling that also has undefined β-catenin-independent actions. Here we report for the first time that a proportion of DKK-1 locates within the nucleus of healthy small intestine and colon mucosa, and of CRC cells at specific chromatin sites of active transcription. Moreover, we show that DKK-1 regulates several cancer-related genes including the cancer stem cell marker aldehyde dehydrogenase 1A1 (ALDH1A1) and Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), which are involved in detoxification of chemotherapeutic agents. Nuclear DKK-1 expression is lost along CRC progression; however, it remains high in a subset (15%) of CRC patients (n = 699) and associates with decreased progression-free survival (PFS) after chemotherapy administration and overall survival (OS) [adjusted HR, 1.65; 95% confidence interval (CI), 1.23-2.21; P = 0.002)]. Overexpression of ALDH1A1 and REPS2 associates with nuclear DKK-1 expression in tumors and correlates with decreased OS (P = 0.001 and 0.014) and PFS. In summary, our findings demonstrate a novel location of DKK-1 within the cell nucleus and support a role of nuclear DKK-1 as a predictive biomarker of chemoresistance in colorectal cancer.
Further info: Oncotarget
Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment.
Sainz B Jr, Alcala S, Garcia E, Sanchez-Ripoll Y, Azevedo MM, Cioffi M, Tatari M, Miranda-Lorenzo I, Hidalgo M, Gomez-Lopez G, Cañamero M, Erkan M, Kleeff J, García-Silva S, Sancho P, Hermann PC, Heeschen C.
Gut 2015 Apr 3. pii: gutjnl-2014-308935. doi: 10.1136/gutjnl-2014-308935.
OBJECTIVES:The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN:Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS:We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-β1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS:Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.
Further info: Gut
The contribution of cohesin-SA1 to gene expression and chromatin architecture in two murine tissues.
Ana Cuadrado, Silvia Remeseiro, Osvaldo Graña, David G. Pisano and Ana Losada.
Nucleic Acids Research 2015 March 3;doi: 10.1093/nar/gkv144
Cohesin, which in somatic vertebrate cells consists of SMC1, SMC3, RAD21 and either SA1 or SA2, mediates higher-order chromatin organization. To determine how cohesin contributes to the establishment of tissue-specific transcriptional programs, we compared genome-wide cohesin distribution, gene expression and chromatin architecture in cerebral cortex and pancreas from adult mice. More than one third of cohesin binding sites differ between the two tissues and these show reduced overlap with CCCTC-binding factor (CTCF) and are enriched at the regulatory regions of tissue-specific genes. Cohesin/CTCF sites at active enhancers and promoters contain, at least, cohesin-SA1. Analyses of chromatin contacts at the Protocadherin (Pcdh) and Regenerating islet-derived (Reg) gene clusters, mostly expressed in brain and pancreas, respectively, revealed remarkable differences that correlate with the presence of cohesin. We could not detect significant changes in the chromatin contacts at the Pcdh locus when comparing brains from wild-type and SA1 null embryos. In contrast, reduced dosage of SA1 altered the architecture of the Reg locus and decreased the expression of Reg genes in the pancreas of SA1 heterozygous mice. Given the role of Reg proteins in inflammation, such reduction may contribute to the increased incidence of pancreatic cancer observed in these animals.
Further info: NAR
Telomerase expression confers cardioprotection in the adult mouse heart after acute myocardial infarction.
Bär C, Bernardes de Jesus B, Serrano R, Tejera A, Ayuso E, Jimenez V, Formentini I, Bobadilla M, Mizrahi J, de Martino A, Gomez G, Pisano D, Mulero F, Wollert KC, Bosch F, Blasco MA.
Nature Communications 2014 Dec 18;5:5863.
Coronary heart disease is one of the main causes of death in the developed world, and treatment success remains modest, with high mortality rates within 1 year after myocardial infarction (MI). Thus, new therapeutic targets and effective treatments are necessary. Short telomeres are risk factors for age-associated diseases, including heart disease. Here we address the potential of telomerase (Tert) activation in prevention of heart failure after MI in adult mice. We use adeno-associated viruses for cardiac-specific Tert expression. We find that upon MI, hearts expressing Tert show attenuated cardiac dilation, improved ventricular function and smaller infarct scars concomitant with increased mouse survival by 17% compared with controls. Furthermore, Tert treatment results in elongated telomeres, increased numbers of Ki67 and pH3-positive cardiomyocytes and a gene expression switch towards a regeneration signature of neonatal mice. Our work suggests telomerase activation could be a therapeutic strategy to prevent heart failure after MI.
Further info: Nature Communications
Inhibition of De Novo NAD+ Synthesis by Oncogenic URI Causes Liver Tumorigenesis through DNA Damage.
Krishna S. Tummala, Ana L. Gomes, Mahmut Yilmaz, Osvaldo Graña, Latifa Bakiri, Isabel Ruppen, Pilar Ximénez-Embún, Vinayata Sheshappanavar, Manuel Rodriguez-Justo, David G Pisano ,Erwin F. Wagner and Nabil Djouder.
Cancer Cell 2014 Dec 8;26(6):826-39. doi: 10.1016/j.ccell.2014.10.002.
Molecular mechanisms responsible for hepatocellular carcinoma (HCC) remain largely unknown. Using genetically engineered mouse models, we show that hepatocyte-specific expression of unconventional prefoldin RPB5 interactor (URI) leads to a multistep process of HCC development, whereas its genetic reduction in hepatocytes protects against diethylnitrosamine (DEN)-induced HCC. URI inhibits aryl hydrocarbon (AhR)- and estrogen receptor (ER)-mediated transcription of enzymes implicated in L-tryptophan/kynurenine/nicotinamide adenine dinucleotide (NAD+) metabolism, thereby causing DNA damage at early stages of tumorigenesis. Restoring NAD+ pools with nicotinamide riboside (NR) prevents DNA damage and tumor formation. Consistently, URI expression in human HCC is associated with poor survival and correlates negatively with L-tryptophan catabolism pathway. Our results suggest that boosting NAD+ can be prophylactic or therapeutic in HCC.
Further info: Cancer Cell
Identification of TERRA locus unveils a telomere protection role through association to nearly all chromosomes.
Isabel López de Silanes, Osvaldo Graña, Maria Luigia De Bonis, Orlando Dominguez, David G Pisano and Maria A Blasco.
Nature Communications 2014 Sep 3;5:4723.
Telomeric RNAs (TERRAs) are UUAGGG repeat-containing RNAs that are transcribed from the subtelomere towards the telomere. The precise genomic origin of TERRA has remained elusive. Using a whole-genome RNA-sequencing approach, we identify novel mouse transcripts arising mainly from the subtelomere of chromosome 18, and to a lesser extend chromosome 9, that resemble TERRA in several key aspects. Those transcripts contain UUAGGG-repeats and are heterogeneous in size, fluctuate in abundance in a TERRA-like manner during the cell cycle, are bound by TERRA RNA-binding proteins and are regulated in a manner similar to TERRA in response to stress and the induction of pluripotency. These transcripts are also found to associate with nearly all chromosome ends and downregulation of the transcripts that originate from chromosome 18 causes a reduction in TERRA abundance. Interestingly, downregulation of either chromosome 18 transcripts or TERRA results in increased number of telomere dysfunction-induced foci, suggesting a protective role at telomeres.
Further info: Nature Communications
Sox4 Links Tumor Suppression to Accelerated Aging in Mice by Modulating Stem Cell Activation.
Miguel Foronda, Paula Martínez, Stefan Schoeftner, Gonzalo Gómez-López, Ralph Schneider, Juana M. Flores, David G. Pisano, Maria A. Blasco.
Cell Reports, http://dx.doi.org/10.1016/j.celrep.2014.06.031
Sox4 expression is restricted in mammals to embryonic structures and some adult tissues, such as lymphoid organs, pancreas, intestine, and skin. During embryogenesis, Sox4 regulates mesenchymal and neural progenitor survival, as well as lymphocyte and myeloid differentiation, and contributes to pancreas, bone, and heart development. Aberrant Sox4 expression is linked to malignant transformation and metastasis in several types of cancer. To understand the role of Sox4 in the adult organism, we first generated mice with reduced whole-body Sox4 expression. These mice display accelerated aging and reduced cancer incidence. To specifically address a role for Sox4 in adult stem cells, we conditionally deleted Sox4 (Sox4cKO) in stratified epithelia. Sox4cKO mice show increased skin stem cell quiescence and resistance to chemical carcinogenesis concomitantly with downregulation of cell cycle, DNA repair, and activated hair follicle stem cell pathways. Altogether, these findings highlight the importance of Sox4 in regulating adult tissue homeostasis and cancer.
Further info: Cell Reports
Fos-dependent induction of Chk1 protects osteoblasts from replication stress.
Schulze J, Lopez-Contreras AJ, Uluçkan Ö, Graña-Castro O, Fernandez-Capetillo O, Wagner EF.
Cell Cycle 2014;13(12):1980-6.
Stable Fos expression in the osteoblast lineage results in the development of osteosarcomas (OS) in mice, yet the underlying mechanisms are poorly understood. Using a genetic system in which Fos expression can be induced in osteoblasts in a doxycycline-dependent manner and through subsequent RNA sequencing and gene set enrichment analysis, we were able to identify novel transcriptional targets of Fos in osteoblasts. These included a distinct activation of cellular response toward replication stress (RS), exemplified by a Fos-dependent induction of the RS-suppressing Chk1 kinase. Importantly, Fos expression protects osteoblasts from RS and DNA damage likely through upregulation of Chk1 and facilitates transformation by Ras/E1A oncogenes. These data reveal a novel function of Fos in safeguarding genome stability during replication, which is particularly relevant in conditions of oncogene-induced S-phase entry.
Further info:Cell Cycle
Lineage-restricted function of the pluripotency factor NANOG in stratified epithelia.
Piazzolla D, Palla AR, Pantoja C, Cañamero M, de Castro IP, Ortega S, Gomez-Lopez G, Dominguez O, Megías D, Roncador G, Luque-Garcia JL, Fernandez-Tresguerres B, Fernandez AF, Fraga MF, Rodriguez-Justo M, Manzanares M, Sánchez-Carbayo M, García-Pedrero JM, Rodrigo JP, Malumbres M, Serrano M.
Nat Commun. 2014 Jun 30;5:4226.
NANOG is a pluripotency transcription factor in embryonic stem cells; however, its role in adult tissues remains largely unexplored. Here we show that mouse NANOG is selectively expressed in stratified epithelia, most notably in the oesophagus where the Nanog promoter is hypomethylated. Interestingly, inducible ubiquitous overexpression of NANOG in mice causes hyperplasia selectively in the oesophagus, in association with increased cell proliferation. NANOG transcriptionally activates the mitotic programme, including Aurora A kinase (Aurka), in stratified epithelia, and endogenous NANOG directly binds to the Aurka promoter in primary keratinocytes. Interestingly, overexpression of Nanog or Aurka in mice increased proliferation and aneuploidy in the oesophageal basal epithelium. Finally, inactivation of NANOG in cell lines from oesophageal or head and neck squamous cell carcinomas (ESCCs or HNSCCs, respectively) results in lower levels of AURKA and decreased proliferation, and NANOG and AURKA expression are positively correlated in HNSCCs. Together, these results indicate that NANOG has a lineage-restricted mitogenic function in stratified epithelia.
Further info: Nature Communications
RAB7 controls melanoma progression by exploiting a lineage-specific wiring of the endolysosomal pathway.
Direna Alonso-Curbelo, Erica Riveiro-Falkenbach, Eva Pérez-Guijarro, Metehan Cifdaloz, Panagiotis Karras, Lisa Osterloh, Diego Megías, Estela Cañón, Tonantzin G. Calvo, David Olmeda, Gonzalo Gomez-Lopez, Víctor Javier Sánchez-Arévalo Lobo, Osvaldo Graña,Agnieszka Checinska, David G. Pisano, Hao-Wei Wang, Pablo Ortiz-Romero, Damià Tormo, José L Rodríguez-Peralto, Johanna A. Joyce, María S. Soengas.
Cancer Cell 2014 June 27
Although common cancer hallmarks are well established, lineage-restricted oncogenes remain less understood. Here, we report an inherent dependency of melanoma cells on the small GTPase RAB7, identified within a lysosomal gene cluster that distinguishes this malignancy from over 35 tumor types. Analyses in human cells, clinical specimens, and mouse models demonstrated that RAB7 is an early-induced melanoma driver whose levels can be tuned to favor tumor invasion, ultimately defining metastatic risk. Importantly, RAB7 levels and function were independent of MITF, the best-characterized melanocyte lineage-specific transcription factor. Instead, we describe the neuroectodermal master modulator SOX10 and the oncogene MYC as RAB7 regulators. These results reveal a unique wiring of the lysosomal pathway that melanomas exploit to foster tumor progression.
Further info: Cancer Cell
MicroRNA expression signatures for the prediction of BRCA1/2-mutation associated hereditary breast cancer in paraffin-embedded formalin-fixed breast tumors.
Tanic M, Yanowski K, Gomez-Lopez G, Rodriguez-Pinilla Socorro M, Marquez-Rodas I, Osorio A, Pisano DG, Martinez-Delgado B, Benítez J.
Int J Cancer. 2014 Jun 11. doi: 10.1002/ijc.29021.
Screening for germline mutations in BRCA1 and BRCA2 genes is indicated for breast cancer patients from high-risk breast cancer families, and influences both treatment options and clinical management. However, only 25% of selected patients test positive for BRCA1/2 mutation, indicating that additional diagnostic biomarkers are necessary. We analyzed 124 FFPE tumor samples from patients with hereditary (104) and sporadic (20) invasive breast cancer, divided into two series (A and B). Microarray expression profiling of 829 human miRNAs was performed on 76 samples (series A), and bioinformatics tool Prophet was used to develop and test a microarray classifier. Samples were stratified into a training set (n=38), for microarray classifier generation and a test set (n=38) for signature validation. A 35-miRNA microarray classifier was generated for the prediction of BRCA1/2 mutation status with a reported 95% (95% CI: 0.88 -1), and 92% (95% CI: 0.84 -1) accuracy in the training and the test set, respectively. Differential expression of 12 miRNAs between BRCA1/2 mutation-carriers versus non-carriers was validated by qPCR in an independent tumor Series B (n=48). Logistic regression model based on the expression of 6 miRNAs (miR-142-3p, miR-505*, miR-1248, miR-181a-2*, miR-25* and miR-340*) discriminated between tumors from BRCA1/2 mutation-carriers and non-carriers with 92% (95% CI: 0.84 -0.99) accuracy. In conclusion, we identified miRNA expression signatures predictive of BRCA1/2 mutation status in routinely available FFPE breast tumor samples, which may be useful to complement current patient selection criteria for gene testing by identifying individuals with high likelihood of being BRCA1/2 mutation carriers.
Further info: International Journal of Cancer
The Epstein Barr-encoded BART-6-3p microRNA affects regulation of cell growth and immuno response in Burkitt lymphoma.
Ambrosio MR, Navari M, Di Lisio L, Leon EA, Onnis A, Gazaneo S, Mundo L, Ulivieri C, Gomez G, Lazzi S, Piris MA, Leoncini L, De Falco G.
Infect Agent Cancer. 2014 Apr 14;9(1):12.
BACKGROUND:Burkitt lymphoma is an aggressive B-cell lymphoma presenting in three clinical forms: endemic, sporadic and immunodeficiency-associated. More than 90% of endemic Burkitt lymphoma carry latent Epstein-Barr virus, whereas only 20% of sporadic Burkitt lymphoma are associated with Epstein-Barr infection. Although the Epstein-Barr virus is highly related with the endemic form, how and whether the virus participates in its pathogenesis remains to be fully elucidated. In particular, the virus may impair cellular gene expression using its own encoded microRNAs.
METHODS:Using microRNA profiling we compared Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for both cellular and viral microRNA. The array results were validated by qRT-PCR, and potential targets of viral microRNAs were then searched by bioinformatic predictions, and classified in functional categories, according to the Gene Ontology. Our findings were validated by in vitro functional studies and by immunohistochemistry on a larger series of cases.
RESULTS:We showed that a few cellular microRNAs are differentially expressed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases, and identified a subset of viral microRNAs expressed in Epstein-Barr-positive Burkitt lymphomas. Of these, we characterized the effects of viral BART6-3p on regulation of cellular genes. In particular, we analyzed the IL-6 receptor genes (IL-6Ralpha and IL-6ST), PTEN and WT1 expression for their possible relevance to Burkitt lymphoma. By means of immunohistochemistry, we observed a down-regulation of the IL-6 receptor and PTEN specifically in Epstein-Barr-positive Burkitt lymphoma cases, which may result in the impairment of key cellular pathways and may contribute to malignant transformation. On the contrary, no differences were observed between Epstein-Barr-positive and Epstein-Barr-negative Burkitt lymphoma cases for WT1 expression.
CONCLUSIONS:Our preliminary results point at an active role for the Epstein-Barr virus in Burkitt lymphomagenesis and suggest new possible mechanisms used by the virus in determining dysregulation of the host cell physiology.
Further info: Infect Agent Cancer
Fos-dependent induction of Chk1 protects osteoblasts from replication stress.
Schulze J, López-Contreras AJ, Uluçkan O, Graña O, Fernández-Capetillo O, Wagner EF.
Cell Cycle. 13; 12. 2014
Stable Fos expression in the osteoblast lineage results in the development of osteosarcomas (OS) in mice, yet the underlying mechanisms are poorly understood. Using a genetic system in which Fos expression can be induced in osteoblasts in a Doxycycline-dependent manner and through subsequent RNA sequencing and gene set enrichment analysis, we were able to identify novel transcriptional targets of Fos in osteoblasts. These included a distinct activation of cellular response toward replication stress (RS), exemplified by a Fos-dependent induction of the RS-suppressing Chk1 kinase. Importantly, Fos expression protects osteoblasts from RS and DNA damage through upregulation of Chk1 and facilitates transformation by Ras/E1A oncogenes. These data reveal a novel function of Fos in safeguarding genome stability during replication, which is particularly relevant in conditions of oncogene-induced S-phase entry.
Further info: Cell Cycle
PLCG1 mutations in cutaneous T-cell lymphomas.
Vaqué JP, Gomez-Lopez G, Monsálvez V, Varela I, Martínez N, Pérez C, Domínguez O, Graña O, Rodríguez-Peralto JL, Rodríguez-Pinilla SM, González-Vela C, Rubio-Camarillo M, Martín-Sánchez E, Pisano DG, Papadavid E, Papadaki T, Requena L, García-Marco JA, Méndez M, Provencio M, Hospital M, Suárez-Massa D, Postigo C, San Segundo D, López-Hoyos M, Ortiz-Romero PL, Piris MA, Sánchez-Beato M.
Blood. 2014 Feb 4
Cutaneous T-cell lymphoma (CTCL) is a heterogeneous group of primary cutaneous T-cell lymphoproliferative processes, mainly composed of mycosis fungoides and Sézary syndrome, and whose aggressive forms lack effective treatment. The molecular pathogenesis of CTCL is largely unknown, although neoplastic cells show increased signaling from T-cell receptors (TCR). DNAs from 11 CTCL patients, both normal and tumoral, were target-enriched and sequenced by massive parallel sequencing for a selection of 524 TCR-signaling related genes. Identified variants were validated by capillary sequencing. Multiple mutations were found that affected several signaling pathways like TCR, NFKB or JAK-STAT, but PLCG1 was found to be mutated in three samples, two of which featured a redundant mutation (c.1034T>C, S345F) in exon 11 that affects the PLCx protein catalytic domain. This mutation was further analyzed by qPCR-genotyping in a new cohort of 42 CTCL patients, where it was found in 19% of samples. Immunohistochemical analysis for NFAT showed that PLCG1-mutated cases exhibited strong NFAT nuclear immunostaining. Functional studies demonstrated that PLCG1 mutants elicited increased downstream signaling towards NFAT activation, and inhibition of this pathway resulted in reduced CTCL cell proliferation and cell viability. Thus, increased proliferative and survival mechanisms in CTCL may partially depend on the acquisition of somatic mutations in PLCG1 and other genes that are essential for normal T-cell differentiation.
Further info: Blood
Transcriptome characterization by RNA sequencing identifies a major molecular and clinical subdivision in chronic lymphocytic leukemia.
Ferreira PG, Jares P, Rico D, Gomez-Lopez G, Martínez-Trillos A, Villamor N, Ecker S, González-Pérez A, Knowles DG, Monlong J, Johnson R, Quesada V, Gouin A, Djebali S, López-Guerra M, Colomer D, Royo C, Cazorla M, Pinyol M, Clot G, Aymerich M, Rozman M, Kulis M, Tamborero D, Papasaikas P, Blanc J, Gut M, Gut I, Puente XS, Pisano DG, Martin-Subero JI, López-Bigas N, López-Guillermo A, Valencia A, López-Otín C, Campo E, Guigo R.
Genome Res. 2013 Nov 21.
Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein coding genes, non-coding RNAs and pseudogenes. Transposable elements are globally de-repressed in CLL cells. In addition, two thousand genes - most of which are not differentially expressed - exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome and ribosome were among the most downregulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.
Further info: Genome Research
Programmed Cell Senescence during Mammalian Embryonic Development.
Muñoz-Espín D, Cañamero M, Maraver A, Gomez-Lopez G, Contreras J, Murillo-Cuesta S, Rodríguez-Baeza A, Varela-Nieto I, Ruberte J, Collado M, Serrano M.
Cell. 2013 Nov 13. doi:pii: S0092-8674(13)01295-6. 10.1016/j.cell.2013.10.019.
Cellular senescence disables proliferation in damaged cells, and it is relevant for cancer and aging. Here, we show that senescence occurs during mammalian embryonic development at multiple locations, including the mesonephros and the endolymphatic sac of the inner ear, which we have analyzed in detail. Mechanistically, senescence in both structures is strictly dependent on p21, but independent of DNA damage, p53, or other cell-cycle inhibitors, and it is regulated by the TGF-β/SMAD and PI3K/FOXO pathways. Developmentally programmed senescence is followed by macrophage infiltration, clearance of senescent cells, and tissue remodeling. Loss of senescence due to the absence of p21 is partially compensated by apoptosis but still results in detectable developmental abnormalities. Importantly, the mesonephros and endolymphatic sac of human embryos also show evidence of senescence. We conclude that the role of developmentally programmed senescence is to promote tissue remodeling and propose that this is the evolutionary origin of damage-induced senescence.
Further info: Cell
CSF3R T618I co-occurs with mutations of splicing and epigenetic genes and with a new PIM3 truncated fusion gene in chronic neutrophilic leukemia.
Menezes J, Makishima H, Gomez I, Acquadro F, Gomez-Lopez G, Graña O, Dopazo A, Alvarez S, Trujillo M, Pisano DG, Maciejewski JP, Cigudosa JC.
Blood Cancer J. 2013 Nov 8;3:e158. doi: 10.1038/bcj.2013.55.
Mutations in CSF3R have been recently defined as the common genetic event in patients with myeloid neoplasms, including the rare entity known as chronic neutrophilic leukemia (CNL), becoming a potentially useful biomarker for diagnosing and therapy target. CSF3R encodes the transmembrane receptor for granulocyte colony-stimulating factor (G-CSF; CSF3), which provides the proliferative and survival signal for granulocytes and also contributes to their differentiation and function. Although there are several studies on massive next-generation sequencing of myeloid disorders, not a single comprehensive study has been reported in CNL. Here, we used whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify new candidate genes to the disease pathogenesis of an index CNL patient.
Further info: Blood Cancer Journal
ASXL1, TP53 and IKZF3 mutations are present in the chronic phase and blast crisis of chronic myeloid leukemia.
Menezes J, Salgado RN, Acquadro F, Gomez-Lopez G, Carralero MC, Barroso A, Mercadillo F, Espinosa-Hevia L, Talavera-Casañas JG, Pisano DG, Alvarez S, Cigudosa JC.
Blood Cancer J. 2013 Nov 8;3:e157. doi: 10.1038/bcj.2013.54.
The mechanism of transformation from chronic phase (CP) to blast crisis (BC) chronic myeloid leukemia (CML) is heterogeneous and poorly understood.1 The most frequently genetic aberration observed at this advanced stage includes a second Philadelphia (Ph) chromosome, trisomy 8, isochromosome 17 and trisomy 19, alone or in various combinations and complex aberrations. Clonal cytogenetic evolution appears to be the most consistent predictor of blastic transformation, present in up to 80% of patients, until now. At present, little is known about the molecular mechanisms underlying the mutational profilling at CP-CML disease progression and only limited changes occuring during clonal evolution at BC have been described. We read with great interest a series of manuscripts reporting somatic mutations in patients with CML in both the CP and BC. Mutations in genes such as TP53, NPM1, IKZF1, RUNX1 and ASXL1 have been described in CML progression. However, all these studies have analyzed a limited number of genes and mainly focused on BC phase. Interestingly, Soverini et al. performed a massive parallel sequencing at three different stages (diagnosis, major molecular response and disease progression) of a patient who developed a lymphoid BC. In this study, IDH2 R140Q was detected in a very low number of cases, and the mutation was only observed in BC.
Further info: Blood Cancer Journal
ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.
Carlos AR, Escandell JM, Kotsantis P, Suwaki N, Bouwman P, Badie S, Folio C, Benitez J, Gomez-Lopez G, David G Pisano, Jonkers J, Tarsounas M.
Nat Commun. 2013 Oct 28;4:2697. doi: 10.1038/ncomms3697.
ARF is a tumour suppressor activated by oncogenic stress, which stabilizes p53. Although p53 is a key component of the response to DNA damage, a similar function for ARF has not been ascribed. Here we show that primary mouse and human cells lacking the tumour suppressor BRCA2 accumulate DNA damage, which triggers checkpoint signalling and ARF activation. Furthermore, senescence induced by Brca2 deletion in primary mouse and human cells is reversed by the loss of ARF, a phenotype recapitulated in cells lacking RAD51. Surprisingly, ARF is not necessary for p53 accumulation per se but for altering the spectrum of genes activated by this transcription factor. Specifically, ARF enables p53 transcription of Dusp4 and Dusp7, which encode a pair of phosphatases known to inactivate the MAP kinases ERK1/2. Our results ascribe a previously unanticipated function to the ARF tumour suppressor in genome integrity, controlled by replicative stress and ATM/ATR-dependent checkpoint responses.
Further info: Nature Communications
MicroRNA-based molecular classification of non-BRCA1/2 hereditary breast tumours.
M Tanic, Eduardo Andrés León, S M Rodriguez-Pinilla, I Marquez-Rodas, M Cebollero-Presmanes, V Fernandez, A Osorio, J Benítez and B Martinez-Delgado.
British Journal of Cancer 2013 Oct 8;doi:10.1038/bjc.2013.612
Hereditary breast cancer comprises 5–10% of all breast cancers. Mutations in two high-risk susceptibility genes, BRCA1 and BRCA2, along with rare intermediate-risk genes and common low-penetrance alleles identified, altogether explain no more than 45% of the high-risk breast cancer families, although the majority of cases are unaccounted for and are designated as BRCAX tumours. Micro RNAs have called great attention for classification of different cancer types and have been implicated in a range of important biological processes and are deregulated in cancer pathogenesis.
Further info: British Journal of Cancer
Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy
Cristina Balbás-Martínez, Ana Sagrera, Enrique Carrillo-de-Santa-Pau, Julie Earl, Mirari Márquez, Miguel Vazquez, Eleonora Lapi, Francesc Castro-Giner, Sergi Beltran, Mònica Bayés, Alfredo Carrato, Juan C Cigudosa, Orlando Domínguez, Marta Gut, Jesús Herranz, Núria Juanpere, Manolis Kogevinas, Xavier Langa, Elena López-Knowles, José A Lorente, Josep Lloreta, David G Pisano, Laia Richart, Daniel Rico, Rocío N Salgado, Adonina Tardón, Stephen Chanock, Simon Heath, Alfonso Valencia, Ana Losada, Ivo Gut, Núria Malats and Francisco X Real.
Nature Genetics (2013) doi:10.1038/ng.2799
Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management1. A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy.
Further info: Nature Genetics
Exome sequencing reveals novel and recurrent mutations with clinical impact in blastic plasmacytoid dendritic cell neoplasm.
Menezes J, Acquadro F, Wiseman M, Gonzalo Gómez-López, Salgado RN, Talavera-Casañas JG, Buño I, Cervera JV, Montes-Moreno S, Hernández-Rivas JM, Ayala R, Calasanz MJ, Larrayoz MJ, Brichs LF, Gonzalez-Vicent M, Pisano DG, Piris MA, Alvarez S, Cigudosa JC.
Leukemia. 2013 Sep 27. doi: 10.1038/leu.2013.283
Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a very rare disease that currently lacks genomic and genetic biomarkers to assist in its clinical management. We performed whole-exome-sequencing (WES) of three BPDCN cases. Based on these data, we designed a resequencing approach to identify mutations in 38 selected genes in 25 BPDCN samples. WES revealed 37 to 99 deleterious gene mutations per exome with no common affected genes between patients, but with clear overlap in terms of molecular and disease pathways (hematological and dermatological disease). We identified for the first time deleterious mutations in IKZF3, HOXB9, UBE2G2 and ZEB2 in human leukemia. Target-sequencing identified 29 recurring genes, ranging in prevalence from 36% for previously known genes, such as TET2, to 12-16% for newly identified genes, such as IKZF3 or ZEB2. Half of the tumors had mutations affecting either the DNA methylation or chromatin remodeling pathways. The clinical analysis revealed that patients with mutations in DNA methylation pathway had a significantly reduced overall survival (P=0.047). We provide the first mutational profiling of BPDCN. The data support the current WHO classification of the disease as a myeloid disorder and provide a biological rationale for the incorporation of epigenetic therapies for its treatment.
Further info: Leukemia journal
Reprogramming in vivo produces teratomas and iPS cells with totipotency features.
Abad M, Mosteiro L, Pantoja C, Cañamero M, Rayon T, Ors I, Graña O, Megías D, Domínguez O, Martínez D, Manzanares M, Ortega S, Serrano M.
Nature 2013 Sep 11. doi: 10.1038/nature12586.
Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.
Further info: Nature
RAP1 protects from obesity through its extratelomeric role regulating gene expression.
Paula Martínez, Gonzalo Gómez-López, Fernando García, Evi Mercken, Sarah Mitchell, Juana M. Flores, Rafael de Cabo, Maria A. Blasco.
Cell Reports (2013). doi: 10.1016/j.celrep.2013.05.030
RAP1 is part of shelterin, the protective complex at telomeres. RAP1 also binds along chromosome arms, where it is proposed to regulate gene expression. To investigate the nontelomeric roles of RAP1 in vivo, we generated a RAP1 whole-body knockout mouse. These mice show early onset of obesity, which is more severe in females than in males. Rap1-deficient mice show accumulation of abdominal fat, hepatic steatosis, and high-fasting plasma levels of insulin, glucose, cholesterol, and alanine aminotransferase. Gene expression analyses of liver and visceral white fat from Rap1-deficient mice before the onset of obesity show deregulation of metabolic programs, including fatty acid, glucose metabolism, and PPARα signaling. We identify Pparα and Pgc1α as key factors affected by Rap1 deletion in the liver. We show that RAP1 binds to Pparα and Pgc1α loci and modulates their transcription. These findings reveal a role for a telomere-binding protein in the regulation of metabolism.
Further info: Cell Reports
INK4a/ARF limits the expansion of cells suffering from replication stress.
Monasor A, Murga M, Lopez-Contreras A, Navas C, Gómez G, Pisano D, Fernandez-Capetillo O.
Cell Cycle 2013; 12:(12).
Replication stress (RS) is a source of DNA damage that has been linked to cancer and aging, which is suppressed by the ATR kinase. In mice, reduced ATR levels in a model of the ATR-Seckel syndrome lead to RS and accelerated aging. Similarly, ATR-Seckel embryonic fibroblasts (MEF) accumulate RS and undergo cellular senescence. We previously showed that senescence of ATR-Seckel MEF cannot be rescued by p53-deletion. Here, we show that the genetic ablation of the INK4a/Arf locus fully rescues senescence on ATR mutant MEF, but also that induced by other conditions that generate RS such as low doses of hydroxyurea or ATR inhibitors. In addition, we show that a persistent exposure to RS leads to increased levels of INK4a/Arf products, revealing that INK4a/ARF behaves as a bona fide RS checkpoint. Our data reveal an unknown role for INK4a/ARF in limiting the expansion of cells suffering from persistent replication stress, linking this well-known tumor suppressor to the maintenance of genomic integrity.
Further info: Cell Cycle
New Insights into FoxE1 Functions: Identification of Direct FoxE1 Targets in Thyroid Cells.
Fernández LP, López-Márquez A, Martínez AM, Gómez-López G, Santisteban P.
PLoS One. 2013 May 13;8(5):e62849.
FoxE1 is a thyroid-specific forkhead transcription factor essential for thyroid gland development, as well as for the maintenance of the thyroid differentiated state in adults. FoxE1 recognizes and binds to a short DNA sequence present in thyroglobulin (Tg) and thyroperoxidase (Tpo) promoters, but FoxE1 binding to regulatory regions other than Tg and Tpo promoters remains almost unexplored. Improving knowledge of the regulatory functions of FoxE1 is necessary to clarify its role in endocrine syndromes and cancer susceptibility. In order to further investigate downstream FoxE1 targets, we performed a genome-wide expression screening after knocking-down FoxE1 and obtained new insights into FoxE1 transcriptional networks in thyroid follicular cells. After validation, we confirmed Adamts9, Cdh1, Duox2 and S100a4 as upregulated genes and Casp4, Creld2, Dusp5, Etv5, Hsp5a, Nr4a2 and Tm4sf1 as downregulated genes when FoxE1 was silenced. In promoter regions of putative FoxE1-regulated genes and also in the promoters of the classical thyroid genes Nis, Pax8 and Titf1, we performed an in silico search of the FoxE1 binding motif that was in close proximity to the NF1/CTF binding sequence, as previously described for other forkhead factors. Using chromatin immunoprecipitation we detected specific in vivo FoxE1 binding to novel regulatory regions in two relevant thyroid genes, Nis and Duox2. Moreover, we demonstrated simultaneous binding of FoxE1 and NF1/CTF to the Nis upstream enhancer region, as well as a clear functional activation of the Nis promoter by both transcription factors. In search for potential downstream mediators of FoxE1 function in thyroid cells, we identified two novel direct FoxE1 target genes. To our knowledge, this is the first evidence regarding the implication of Nis and Duox2 in executing the transcriptional program triggered by FoxE1. Furthermore, this study points out the important role of FoxE1 in the regulation of a large number of genes in thyroid cells.
Further info: PLoS One
RUbioSeq: a suite of parallelized pipelines to automate exome variation and bisulfite-seq analyses.
Rubio-Camarillo M, Gómez-López G, Fernández JM, Valencia A, Pisano DG.
Bioinformatics. 2013 Apr 28
RUbioSeq has been developed to facilitate the primary and secondary analysis of resequencing projects by providing an integrated software suite of parallelized pipelines to detect exome variants (SNVs and CNVs) and to perform Bisulfite-seq analyses automatically. RUbioSeq's variant analysis results have been already validated and published. AVAILABILITY: http://rubioseq.sourceforge.net/
Further info: Bioinformatics Journal
ARID1A alterations are associated with FGFR3-wild type, poor-prognosis, urothelial bladder tumors.
Cristina Balbás-Martínez, María Rodríguez-Pinilla, Ariel Casanova, Orlando Domínguez,Pisano DG, Gómez G, Josep Lloreta, José A. Lorente, Nuria Malats, Francisco X. Real.
PLoS One 8(5): e62483. doi:10.1371/journal.pone.0062483
Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological, genetic, and epigenetic levels. Exome sequencing has identified ARID1A as a novel tumor suppressor gene coding for a chromatin remodeling protein that is mutated in UBC. Here, we assess ARID1A alterations in two series of patients with UBC. In the first tumor series, we analyze exons 2–20 in 52 primary UBC and find that all mutant tumors belong to the aggressive UBC phenotype (high grade non-muscle invasive and muscle invasive tumors) (P = 0.05). In a second series (n = 84), we assess ARID1A expression using immunohistochemistry, a surrogate for mutation analysis, and find that loss of expression increases with higher stage/grade, it is inversely associated with FGFR3 overexpression (P = 0.03) but it is not correlated with p53 overexpression (P = 0.30). We also analyzed the expression of cytokeratins in the same set of tumor and find, using unsupervised clustering, that tumors with ARID1A loss of expression are generally KRT5/6-low. In this patient series, loss of ARID1A expression is also associated with worse prognosis, likely reflecting the higher prevalence of losses found in tumors of higher stage and grade. The independent findings in these two sets of patients strongly support the notion that ARID1A inactivation is a key player in bladder carcinogenesis occurring predominantly in FGFR3 wild type tumors.
Further info: PLoS One
Differential Gene Expression of Medullary Thyroid Carcinoma Reveals Specific Markers Associated with Genetic Conditions.
Maliszewska A, Leandro-Garcia LJ, Castelblanco E, Macià A, de Cubas A, Gómez-López G, Inglada-Pérez L, Alvarez-Escolá C, De la Vega L, Letón R, Gómez-Graña A, Landa I, Cascón A, Rodríguez-Antona C, Borrego S, Zane M, Schiavi F, Merante-Boschin I, Pelizzo MR, Pisano DG, Opocher G, Matias-Guiu X, Encinas M, Robledo M.
Am J Pathol. 2012 Nov 28. doi:pii: S0002-9440(12)00824-3. 10.1016/j.ajpath.2012.10.025.
Medullary thyroid carcinoma accounts for 2% to 5% of thyroid malignancies, of which 75% are sporadic and the remaining 25% are hereditary and related to multiple endocrine neoplasia type 2 syndrome. Despite a genotype-phenotype correlation with specific germline RET mutations, knowledge of pathways specifically associated with each mutation and with non-RET-mutated sporadic MTC remains lacking. Gene expression patterns have provided a tool for identifying molecular events related to specific tumor types and to different clinical features that could help identify novel therapeutic targets. Using transcriptional profiling of 49 frozen MTC specimens classified as RET mutation, we identified PROM1, LOXL2, GFRA1, and DKK4 as related to RET(M918T) and GAL as related to RET(634) mutation. An independent series of 19 frozen and 23 formalin-fixed, paraffin-embedded (FFPE) MTCs was used for validation by RT-qPCR. Two tissue microarrays containing 69 MTCs were available for IHC assays. According to pathway enrichment analysis and gene ontology biological processes, genes associated with the MTC(M918T) group were involved mainly in proliferative, cell adhesion, and general malignant metastatic effects and with Wnt, Notch, NFκB, JAK/Stat, and MAPK signaling pathways. Assays based on silencing of PROM1 by siRNAs performed in the MZ-CRC-1 cell line, harboring RET(M918T), caused an increase in apoptotic nuclei, suggesting that PROM1 is necessary for survival of these cells. This is the first report of PROM1 overexpression among primary tumors.
Further info: PubMed
Downregulation of specific miRNAs in hyperdiploid multiple myeloma mimics the oncogenic effect of IgH translocations occurring in the non-hyperdiploid subtype.
Rio-Machin A, Ferreira BI, Henry T, Gómez-López G, Agirre X, Alvarez S, Rodriguez-Perales S, Prosper F, Calasanz MJ, Martínez J, Fonseca R, Cigudosa JC.
Leukemia. 2012 Oct 22. doi: 10.1038/leu.2012.302.
Currently, multiple myeloma (MM) patients are broadly grouped into a non-hyperdiploid (nh-MM) group, highly enriched for IgH translocations, or into a hyperdiploid (h-MM) group, which is typically characterized by trisomies of some odd-numbered chromosomes. We compared the micro RNA (miRNA) expression profiles of these two groups and we identified 16 miRNAs that were downregulated in the h-MM group, relative to the nh-MM group. We found that target genes of the most differentially expressed miRNAs are directly involved in the pathogenesis of MM; specifically, the inhibition of hsa-miR-425, hsa-miR-152 and hsa-miR-24, which are all downregulated in h-MM, leads to the overexpression of CCND1, TACC3, MAFB, FGFR3 and MYC, which are the also the oncogenes upregulated by the most frequent IgH chromosomal translocations occurring in nh-MM. Importantly, we showed that the downregulation of these specific miRNAs and the upregulation of their targets also occur simultaneously in primary cases of h-MM. These data provide further evidence on the unifying role of cyclin D pathways deregulation as the key mechanism involved in the development of both groups of MM. Finally, they establish the importance of miRNA deregulation in the context of MM, thereby opening up the potential for future therapeutic approaches based on this molecular mechanism.
Further info: Leukemia Journal
ChiTaRS: a database of human, mouse and fruit fly chimeric transcripts and RNA-sequencing data..
Frenkel-Morgenstern M, Gorohovski A, Lacroix V, Rogers M, Ibanez K, Boullosa C, Andres Leon Eduardo, Ben-Hur A, Valencia A.
Nucleic Acids Res. 2012 Nov 9.
Chimeric RNAs that comprise two or more different transcripts have been identified in many cancers and among the Expressed Sequence Tags (ESTs) isolated from different organisms; they might represent functional proteins and produce different disease phenotypes. The ChiTaRS database of Chimeric Transcripts and RNA-Sequencing data (http://chitars.bioinfo.cnio.es/) collects more than 16 000 chimeric RNAs from humans, mice and fruit flies, 233 chimeras confirmed by RNA-seq reads and ∼2000 cancer breakpoints. The database indicates the expression and tissue specificity of these chimeras, as confirmed by RNA-seq data, and it includes mass spectrometry results for some human entries at their junctions. Moreover, the database has advanced features to analyze junction consistency and to rank chimeras based on the evidence of repeated junction sites. Finally, 'Junction Search' screens through the RNA-seq reads found at the chimeras' junction sites to identify putative junctions in novel sequences entered by users. Thus, ChiTaRS is an extensive catalog of human, mouse and fruit fly chimeras that will extend our understanding of the evolution of chimeric transcripts in eukaryotes and can be advantageous in the analysis of human cancer breakpoints.
Further info: Nucleic Acids Res. journal
SIRT1 promotes thyroid carcinogenesis driven by PTEN deficiency.
Herranz D, Maraver A, Cañamero M, Gómez-López G, Inglada-Pérez L, Robledo M, Castelblanco E, Matias-Guiu X, Serrano M.
Oncogene. 2012 Sep 17. doi: 10.1038/onc.2012.407.
Current genetic evidence in mice indicates that SIRT1 has potent tumor suppressor activity in a variety of cancer models, with no evidence yet for SIRT1 oncogenic activity in vivo. We report here that transgenic Sirt1 expression is oncogenic in murine thyroid and prostate carcinogenesis initiated by Pten-deficiency. Based on mRNA expression analyses of pre-tumoral murine thyroids, we find that SIRT1 increases c-MYC transcriptional programs. Moreover, we show higher c-MYC protein levels in murine thyroid cancers from Sirt1 transgenic mice. Similarly, SIRT1 is overexpressed in human thyroid cancers and it is positively correlated with c-MYC protein levels. Finally, we show in cultured thyroid cancer cells that SIRT1 stabilizes c-MYC protein. These results implicate SIRT1 as a new candidate target for the treatment of thyroid carcinomas.
Further info: Oncogene journal
Therapeutic Effect of γ-Secretase Inhibition in Kras(G12V)-Driven Non-Small Cell Lung Carcinoma by Derepression of DUSP1 and Inhibition of ERK.
Maraver A, Fernandez-Marcos PJ, Herranz D, Cañamero M, Muñoz-Martin M, Gómez-López G, Mulero F, Megías D, Sanchez-Carbayo M, Shen J, Sanchez-Cespedes M, Palomero T, Ferrando A, Serrano M.
Cancer Cell. 2012 Aug 14;22(2):222-34.
Here, we have investigated the role of the Notch pathway in the generation and maintenance of Kras(G12V)-driven non-small cell lung carcinomas (NSCLCs). We demonstrate by genetic means that γ-secretase and RBPJ are essential for the formation of NSCLCs. Of importance, pharmacologic treatment of mice carrying autochthonous NSCLCs with a γ-secretase inhibitor (GSI) blocks cancer growth. Treated carcinomas present reduced HES1 levels and reduced phosphorylated ERK without changes in phosphorylated MEK. Mechanistically, we show that HES1 directly binds to and represses the promoter of DUSP1, encoding a dual phosphatase that is active against phospho-ERK. Accordingly, GSI treatment upregulates DUSP1 and decreases phospho-ERK. These data provide proof of the in vivo therapeutic potential of GSIs in primary NSCLCs.
Further info: Cancer Cell
Distinct DNA methylomes of newborns and centenarians.
Heyn H, Li N, Ferreira HJ, Moran S, Pisano DG, Gomez A, Diez J, Sanchez-Mut JV, Setien F, Carmona FJ, Puca AA, Sayols S, Pujana MA, Serra-Musach J, Iglesias-Platas I, Formiga F, Fernandez AF, Fraga MF, Heath SC, Valencia A, Gut IG, Wang J, Esteller M.
Proc Natl Acad Sci U S A. 2012 Jun 11.
Human aging cannot be fully understood in terms of the constrained genetic setting. Epigenetic drift is an alternative means of explaining age-associated alterations. To address this issue, we performed whole-genome bisulfite sequencing (WGBS) of newborn and centenarian genomes. The centenarian DNA had a lower DNA methylation content and a reduced correlation in the methylation status of neighboring cytosine-phosphate-guanine (CpGs) throughout the genome in comparison with the more homogeneously methylated newborn DNA. The more hypomethylated CpGs observed in the centenarian DNA compared with the neonate covered all genomic compartments, such as promoters, exonic, intronic, and intergenic regions. For regulatory regions, the most hypomethylated sequences in the centenarian DNA were present mainly at CpG-poor promoters and in tissue-specific genes, whereas a greater level of DNA methylation was observed in CpG island promoters. We extended the study to a larger cohort of newborn and nonagenarian samples using a 450,000 CpG-site DNA methylation microarray that reinforced the observation of more hypomethylated DNA sequences in the advanced age group. WGBS and 450,000 analyses of middle-age individuals demonstrated DNA methylomes in the crossroad between the newborn and the nonagenarian/centenarian groups. Our study constitutes a unique DNA methylation analysis of the extreme points of human life at a single-nucleotide resolution level.
Further info: PNAS
UNG shapes the specificity of AID-induced somatic hypermutation
Pablo Pérez-Durán, Laura Belver, Virginia G. de Yébenes, Pilar Delgado, David G. Pisano, Almudena R. Ramiro.
J Exp Med. 2012 Jun 4.
Secondary diversification of antibodies through somatic hypermutation (SHM) and class switch recombination (CSR) is a critical component of the immune response. Activation-induced deaminase (AID) initiates both processes by deaminating cytosine residues in immunoglobulin genes. The resulting U:G mismatch can be processed by alternative pathways to give rise to a mutation (SHM) or a DNA double-strand break (CSR). Central to this processing is the activity of uracil-N-glycosylase (UNG), an enzyme normally involved in error-free base excision repair. We used next generation sequencing to analyze the contribution of UNG to the resolution of AID-induced lesions. Loss- and gain-of-function experiments showed that UNG activity can promote both error-prone and high fidelity repair of U:G lesions. Unexpectedly, the balance between these alternative outcomes was influenced by the sequence context of the deaminated cytosine, with individual hotspots exhibiting higher susceptibility to UNG-triggered error-free or error-prone resolution. These results reveal UNG as a new molecular layer that shapes the specificity of AID-induced mutations and may provide new insights into the role of AID in cancer development.
Further info: The Journal of Experimental Medicine
New Mutations in Chronic Lymphocytic Leukemia Identified by Target Enrichment and Deep Sequencing.
Elena Doménech, Gonzalo Gómez-López, Daniel Gzlez-Peña, Mar López, Beatriz Herreros, Juliane Menezes, Natalia Gómez-Lozano, Angel Carro, Osvaldo Graña, David G. Pisano, Orlando Domínguez, José A. García-Marco, Miguel A. Piris, Margarita Sánchez-Beato.
PLoS ONE 7(6): e38158. doi:10.1371/journal.pone.0038158
Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.
Further info: PLoS One Journal
The specific contributions of cohesin-SA1 to cohesion and gene expression: Implications for cancer and development.
Cuadrado A, Remeseiro S, Gómez-López G, Pisano DG, Losada A.
Cell Cycle. 2012 Jun 15;11(12).
Besides its well-established role in sister chromatid cohesion, cohesin has recently emerged as major player in the organization of interphase chromatin. Such important function is related to its ability to entrap two DNA segments also in cis, thereby facilitating long-range DNA looping which is crucial for transcriptional regulation, organization of replication factories and V(D)J recombination. Vertebrate somatic cells have two different versions of cohesin, containing Smc1, Smc3, Rad21/Scc1 and either SA1 or SA2, but their functional specificity has been largely ignored. We recently generated a knockout mouse model for the gene encoding SA1, and found that this protein is essential to complete embryonic development. Cohesin-SA1 mediates cohesion at telomeres, which is required for their replication. Telomere defects in SA1- deficient cells provoke chromosome segregation errors resulting in aneuploidy despite robust centromere cohesion. This aneuploidy could explain why heterozygous animals have an earlier onset of tumorigenesis. In addition, the genome-wide distribution of cohesin changes dramatically in the absence of SA1, and the complex shows reduced accumulation at promoters and CTCF sites. As a consequence, gene expression is altered, leading to downregulation of biological processes related to a developmental disorder linked to cohesin function, the Cornelia de Lange Syndrome (CdLS). These results point out a prominent role of cohesin-SA1 in transcriptional regulation, with clear implications in the etiology of CdLS.
Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells.
Sánchez-Bailón MP, Calcabrini A, Gómez-Domínguez D, Morte B, Martín-Forero E, Gómez-López G, Molinari A, Wagner KU, Martín-Pérez J.
Cell Signal. 2012 Jun;24(6):1276-86.
SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.
Further info: http://www.sciencedirect.com/science/article/pii/S0898656812000708
Genome Wide Analysis Of Pax8 Binding Provides New Insights Into Thyroid Functions.
Sergio Ruiz-Llorente, Enrique Carrillo de Santa Pau, Ana Sastre-Perona, Cristina Montero-Conde,Gonzalo Gomez-Lopez,James A Fagin, Alfonso Valencia, David G Pisano, Pilar Santisteban.
BMC Genomics 2012, 13:147 doi:10.1186/1471-2164-13-147.
The transcription factor Pax8 is essential for the differentiation of thyroid cells. However, there are few data on genes transcriptionally regulated by Pax8 other than thyroid-related genes. To better understand the role of Pax8 in the biology of thyroid cells, we obtained transcriptional profiles of Pax8-silenced PCCl3 thyroid cells using whole genome expression arrays and integrated these signals with global cis-regulatory sequencing studies performed by ChIP-Seq analysis.Exhaustive analysis of Pax8 immunoprecipitated peaks demonstrated preferential binding to intragenic regions and CpG-enriched islands, which suggests a role of Pax8 in transcriptional regulation of orphan CpG regions. In addition, ChIP-Seq allowed us to identify Pax8 partners, including proteins involved in tertiary DNA structure (CTCF) and chromatin remodeling (Sp1), and these direct transcriptional interactions were confirmed in vivo. Moreover, both factors modulate Pax8-dependent transcriptional activation of the sodium iodide symporter (Nis) gene promoter. We ultimately combined putative and novel Pax8 binding sites with actual target gene expression regulation to define Pax8-dependent genes. Functional classification suggests that Pax8-regulated genes may be directly involved in important processes of thyroid cell function such as cell proliferation and differentiation, apoptosis, cell polarity, motion and adhesion, and a plethora of DNA/protein-related processes. Our study provides novel insights into the role of Pax8 in thyroid biology, exerted through transcriptional regulation of important genes involved in critical thyrocyte processes. In addition, we found new transcriptional partners of Pax8, which functionally cooperate with Pax8 in the regulation of thyroid gene transcription. Besides, our data demonstrate preferential location of Pax8 in non-promoter CpG regions. These data point to an orphan CpG island-mediated mechanism that represents a novel role of Pax8 in the transcriptional output of the thyrocyte.
Further info: BMC Genomics
Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion.
Ganuza M, Sáiz-Ladera C, Cañamero M, Gómez G, Schneider R, Blasco MA, Pisano D, Paramio JM, Santamaría D, Barbacid M.
EMBO J. 2012 Apr 13. doi: 10.1038/emboj.2012.94.
Cyclin-dependent kinase (Cdk)7, the catalytic subunit of the Cdk-activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)-mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7-expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death.
Further info: EMBO Journal
A unique role of cohesin-SA1 in gene regulation and development.
Remeseiro S, Cuadrado A,Gómez-López G, Pisano DG, Losada A.
EMBO J. 2012 Mar 13. doi: 10.1038/emboj.2012.60.
Vertebrates have two cohesin complexes that consist of Smc1, Smc3, Rad21/Scc1 and either SA1 or SA2, but their functional specificity is unclear. Mouse embryos lacking SA1 show developmental delay and die before birth. Comparison of the genome-wide distribution of cohesin in wild-type and SA1-null cells reveals that SA1 is largely responsible for cohesin accumulation at promoters and at sites bound by the insulator protein CTCF. As a consequence, ablation of SA1 alters transcription of genes involved in biological processes related to Cornelia de Lange syndrome (CdLS), a genetic disorder linked to dysfunction of cohesin. We show that the presence of cohesin-SA1 at the promoter of myc and of protocadherin genes positively regulates their expression, a task that cannot be assumed by cohesin-SA2. Lack of SA1 also alters cohesin-binding pattern along some gene clusters and leads to dysregulation of genes within. We hypothesize that impaired cohesin-SA1 function in gene expression underlies the molecular aetiology of CdLS.
Further info: EMBO Journal
The Tumor Suppressor and Chromatin Remodeling Factor BRG1 Antagonizes Myc Activity and Promotes Cell Differentiation in Human Cancer.
Romero OA, Setien F, John S, Gimenez-Xavier P, Gómez-López G, Pisano D, Condom E, Villanueva A, Hager GL, Sanchez-Cespedes M.
EMBO Mol Med. 2012 Mar 7. doi: 10.1002/emmm.201200236.
BRG1, a member of the SWI/SNF complex, is mutated in cancer, but it is unclear how it promotes tumorigenesis. We report that re-expression of BRG1 in lung cancer cells up-regulates lung-specific transcripts, restoring the gene expression signature of normal lung. Using cell lines from several cancer types we found that those lacking BRG1 do not respond to retinoic acid (RA) or glucocorticoids (GC), while restoration of BRG1 restores sensitivity. Conversely, in SH-SY5Y cells, a paradigm of RA-dependent differentiation, abrogation of BRG1 prevented the response to RA. Further, our data suggest an antagonistic functional connection between BRG1 and MYC, whereby refractoriness to RA and GC by BRG1 inactivation involves deregulation of MYC activity. Mechanistically, some of these effects are mediated by BRG1 binding to MYC and MYC-target promoters. The BRG1-MYC antagonism was also evident in primary tumors. Finally, BRG1 restoration significantly dampened invasion and progression and decreased MYC in lung cancer cells orthotopically implanted in nude mice. Thus, BRG1 inactivation enables cancer cells to sustain undifferentiated gene expression programs and prevent its response to environmental stimuli.
Further info: EMBO Molecular Medicine
Pten Positively Regulates Brown Adipose Function, Energy Expenditure, and Longevity.
Ana Ortega-Molina, Alejo Efeyan, Elena Lopez-Guadamillas, Maribel Muñoz-Martin, Gonzalo Gómez-López, Marta Cañamero, Francisca Mulero, Joaquin Pastor, Sonia Martinez, Eduardo Romanos, M. Mar Gonzalez-Barroso, Eduardo Rial, Angela M. Valverde, James R. Bischoff, Manuel Serrano.
Cell Metabolism Volume 15, Issue 3, 382-394. 2012.
Aging in worms and flies is regulated by the PI3K/Akt/Foxo pathway. Here we extend this paradigm to mammals. Ptentg mice carrying additional genomic copies of Pten are protected from cancer and present a significant extension of life span that is independent of their lower cancer incidence. Interestingly, Ptentg mice have an increased energy expenditure and protection from metabolic pathologies. The brown adipose tissue (BAT) of Ptentg mice is hyperactive and presents high levels of the uncoupling protein Ucp1, which we show is a target of Foxo1. Importantly, a synthetic PI3K inhibitor also increases energy expenditure and hyperactivates the BAT in mice. These effects can be recapitulated in isolated brown adipocytes and, moreover, implants of Ptentg fibroblasts programmed with Prdm16 and Cebpβ form subcutaneous brown adipose pads more efficiently than wild-type fibroblasts. These observations uncover a role of Pten in promoting energy expenditure, thus decreasing nutrient storage and its associated damage.
Further info: http://www.cell.com/cell-metabolism/retrieve/pii/S1550413112000484
MicroRNA signatures in B-cell lymphomas.
L Di Lisio, M Sanchez-Beato, Gómez-López G, M E Rodríguez, S Montes-Moreno, M Mollejo, J Menárquez, M A Martínez, F J Alves,D G Pisano, M A Piris, N Martínez.
Blood Cancer Journal (2012) 2, e57; doi:10.1038/bcj.2012.1
Accurate lymphoma diagnosis, prognosis and therapy still require additional markers. We explore the potential relevance of microRNA (miRNA) expression in a large series that included all major B-cell non-Hodgkin lymphoma (NHL) types. The data generated were also used to identify miRNAs differentially expressed in Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) samples. A series of 147 NHL samples and 15 controls were hybridized on a human miRNA one-color platform containing probes for 470 human miRNAs. Each lymphoma type was compared against the entire set of NHLs. BL was also directly compared with DLBCL, and 43 preselected miRNAs were analyzed in a new series of routinely processed samples of 28 BLs and 43 DLBCLs using quantitative reverse transcription-polymerase chain reaction. A signature of 128 miRNAs enabled the characterization of lymphoma neoplasms, reflecting the lymphoma type, cell of origin and/or discrete oncogene alterations. Comparative analysis of BL and DLBCL yielded 19 differentially expressed miRNAs, which were confirmed in a second confirmation series of 71 paraffin-embedded samples. The set of differentially expressed miRNAs found here expands the range of potential diagnostic markers for lymphoma diagnosis, especially when differential diagnosis of BL and DLBCL is required.
Epstein-Barr virus microRNAs repress BCL6 expression in diffuse large B-cell lymphoma.
D Martín-Pérez, P. Vargiu, S. Montes-Moreno, Eduardo Andres León, S. M. Rodríguez-Pinilla, L. D. Lisio, N. Martínez, R. Rodríguez, M. Mollejo, J. Castellvi, David G. Pisano, M. Sánchez-Beato, and M. A. Piris.
Leukemia 2012 Jan 26.
Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30–40% of all newly diagnosed lymphomas. DLBCL is considered a heterogeneous disease, with some specific clinicopathological variants of DLBCLs being associated with the presence of the EBV.1 EBV is a lymphotropic virus that has been implicated in the development of several lymphoid malignancies, mainly Burkitt lymphoma (BL) and Hodgkin's lymphoma and with low prevalence in DLBCL.1 BCL6 is a key transcriptional repressor during normal B-cell differentiation that has been shown to repress NF-kB in some DLBCLs.2 In some B-cell lymphomas, BCL6 expression was inversely correlated with LMP1 expression, and some evidences suggest that LMP1 can cause downregulation of BCL6(ref. 3), but other possible mechanisms have not been studied. We have found a strong inverse correlation between BCL6 protein expression and EBV infection (P<0.001, Figures 1a and b) in a series of 149 DLBCL samples, where only one out of 34 EBV-positive cases (2.94%) expressed BCL6, although 87 out of 115 EBV-negative cases expressed BCL6 (75.65%). However, this correlation was independent of LMP1 because 54% of EBER-positive samples were LMP1-negative (Spearman, P-value: 0.18). Little is known about the mechanisms that cause the absence of BCL6 in EBV-positive DLBCL; however, the possibility that EBV-encoded miRNAs could contribute to BCL6 repression has never been explored.
Chromatin modifications induced by the AML1-ETO fusion protein reversibly silence its genomic targets through AML1 and Sp1 binding motifs.
Maiques-Diaz A, Chou FS, Wunderlich M, Gómez-López G, Jacinto FV, Rodriguez-Perales S, Larrayoz MJ, Calasanz MJ, Mulloy JC, Cigudosa JC, Alvarez S.
Leukemia. 2012 Jan 13. doi: 10.1038/leu.2011.376.
The AML1-ETO fusion protein, which is present in 10-15% of cases of acute myeloid leukemia, is known to repress myeloid differentiation genes through DNA binding and recruitment of chromatin-modifying proteins and transcription factors in target genes. ChIP-chip analysis of human hematopoietic stem/progenitor cells transduced with the AML1-ETO fusion gene enabled us to identify 1168 AML1-ETO target genes, 103 of which were co-occupied by histone deacetylase 1 (HDAC1) and had lost the hyperacetylation mark at histone H4, and 264 showed a K9 trimethylation at histone H3. Enrichment of genes involved in hematopoietic differentiation and in specific signaling pathways was observed in the presence of these epigenetic modifications associated with an 'inactive' chromatin status. Furthermore, AML1-ETO target genes had a significant correlation between the chromatin marks studied and transcriptional silencing. Interestingly, AML1 binding sites were absent on a large number of selected AML1-ETO promoters and an Sp1 binding site was found in over 50% of them. Reversible silencing induced by the fusion protein in the presence of AML1 and/or Sp1 transcription factor binding site was confirmed. Therefore, this study provides a global analysis of AML1-ETO functional chromatin modifications and identifies the important role of Sp1 in the DNA binding pattern of AML1-ETO, suggesting a role for Sp1-targeted therapy in this leukemia subtype.
Cdc14b regulates mammalian RNA polymerase II and represses cell cycle transcription.
Guillamot M, Manchado E, Chiesa M, Gómez-López G, Pisano DG, Sacristán MP, Malumbres M.
Sci Rep. 2011;1:189. doi:10.1038/srep00189
Cdc14 is an essential phosphatase in yeast but its role in the mammalian cell cycle remains obscure. We report here that Cdc14b-knockout cells display unscheduled induction of multiple cell cycle regulators resulting in early entry into DNA replication and mitosis from quiescence. Cdc14b dephosphorylates Ser5 at the C-terminal domain (CTD) of RNA polymerase II, a major substrate of cyclin-dependent kinases. Lack of Cdc14b results in increased CTD-Ser5 phosphorylation, epigenetic modifications that mark active chromatin, and transcriptional induction of cell cycle regulators. These data suggest a function for mammalian Cdc14 phosphatases in the control of transcription during the cell cycle.
Further info: http://www.nature.com/srep/2011/111212/srep00189/full/srep00189.html
Integration of BRCA1-mediated miRNA and mRNA profiles reveals microRNA regulation of TRAF2 and NFκB pathway.
Tanic M, Zajac M, Gómez-López G, Benítez J, Martínez-Delgado B.
Breast Cancer Res Treat. 2011 Dec 14.
Microarray-based techniques are being useful to obtain miRNA and gene expression signatures associated with different tumors. BRCA1 deregulation is a frequent event in the pathogenesis of breast as well as other cancers. In addition to DNA repair functions of BRCA1, it is involved in a wide range of cellular processes such as cell cycle, chromatin remodeling or transcription. However, the molecular events underlying BRCA1-associated tumorigenesis are still largely unknown. In order to deepen our understanding of BRCA1-associated tumorigenesis, we integrated data from mRNA and miRNA microarray experiments on HCC1937 breast cancer cell line, and the isogenic HCC1937 stably expressing BRCA1, to obtain significant miRNA-mRNA relationships associated with the presence of BRCA1 gene. By using bioinformatic integration of gene and miRNA expression data, we found significant miRNA-gene relationships underlying the array signatures. We additionally evaluated the role of these statistically significant pairs at the biological pathways level and identified MAPK and NF-κB pathways associated with these expression changes. Furthermore, we experimentally validated miRNAs induced by BRCA1 that commonly regulate TRAF2, a key regulator of NF-κB and MAPK pathways. We demonstrate that miR-146a, miR-99b and miR-205, induced in HCC1937 BRCA1-expressing cells, bind and regulate TRAF2 gene. In addition, re-expression of miR-146a, miR-99b or miR-205 in HCC1937 BRCA1-null cells was sufficient to modulate NF-κB activity. Our results demonstrate that integration of mRNA and miRNA associated with BRCA1 expression was useful to discover new miRNA-gene interactions as molecular events underlying BRCA1-mediated tumorigenesis.
Further info: http://www.springerlink.com/content/f225r81l61362173/
Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.
Víctor Quesada, Laura Conde, Neus Villamor, Gonzalo R Ordóñez, Pedro Jares, Laia Bassaganyas, Andrew J Ramsay, Sílvia Beà, Magda Pinyol, Alejandra Martínez-Trillos, Mónica López-Guerra, Dolors Colomer, Alba Navarro, Tycho Baumann, Marta Aymerich, María Rozman, Julio Delgado, Eva Giné, Jesús M Hernández, Marcos González-Díaz, Diana A Puente, Gloria Velasco, José M P Freije, José M C Tubío, Romina Royo, Josep L Gelpí, Modesto Orozco, David G Pisano, Jorge Zamora, Miguel Vázquez, Alfonso Valencia, Heinz Himmelbauer, Mónica Bayés, Simon Heath, Marta Gut, Ivo Gut, Xavier Estivill, Armando López-Guillermo, Xose S Puente, Elías Campo and Carlos López-Otín.
Nat Genet. 2011 Dec 11
Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.
Further info: http://www.nature.com/ng/journal/vaop/ncurrent/full/ng.1032.html
Long-Range Epigenetic Silencing Associates with Deregulation of Ikaros Targets in Colorectal Cancer Cells.
Javierre B, Rodriguez-Ubreva J, Al-Shahrour F, Corominas M, Graña O, Ciudad L, Agirre X, Pisano DG, Valencia A, Roman-Gomez J, Calasanz M, Prosper F, Esteller M, Gonzalez-Sarmiento R, Ballestar E.
Mol Cancer Res.Published on line 7th July 2011.
Transcription factors (TFs) are common targets of epigenetic inactivation in human cancer. Promoter hypermethylation and subsequent silencing of TFs can lead to further deregulation of their targets. In this study, we explored the potential epigenetic deregulation in cancer of Ikaros family genes, which code for essential TFs in cell differentiation and exhibit genetic defects in hematological neoplasias. Unexpectedly, our analysis revealed that Ikaros undergoes very specific promoter hypermethylation in colorectal cancer, including in all the cell lines studied and around 64% of primary colorectal adenocarcinomas, with increasing proportions in advanced Duke's stages. Ikaros hypermethylation occurred in the context of a novel long-range epigenetic silencing (LRES) region. Reintroduction of Ikaros in colorectal cancer cells, ChIP-chip analysis and validation in primary samples led us to identify a number of direct targets that are possibly related with colorectal cancer progression. Our results not only provide the first evidence that LRES can have functional specific effects in cancer, but also identify several deregulated Ikaros targets that may contribute to progression in colorectal adenocarcinoma.
PileLineGUI: a desktop environment for handling genome position files in next-generation sequencing studies.
Hugo López-Fernández, Daniel Glez-Peña, Miguel Reboiro-Jato, Gonzalo Gómez-López, David G Pisano and Florentino Fdez-Riverola.
Nucleic Acids Res. (Web Server Issue). Jul;39 Suppl 2:W562-6. Published on line 6th June, 2011.
Next-generation sequencing (NGS) technologies are making sequence data available on an unprecedented scale. In this context, new catalogs of Single Nucleotide Polymorphism and mutations generated by resequencing studies are usually stored in genome position files (e.g. Variant Call Format, SAMTools pileup, BED, GFF) comprising of large lists of genomic positions, which are difficult to handle by researchers. Here, we present PileLineGUI, a novel desktop application primarily designed for manipulating, browsing and analysing genome position files (GPF), with specific support to somatic mutation finding studies. The developed tool also integrates a new genome browser module specially designed for inspecting GPFs. PileLineGUI is free, multiplatform and designed to be intuitively used by biomedical researchers. PileLineGUI is available at: http://sing.ei.uvigo.es/pileline/pilelinegui.html.
Further info: http://nar.oxfordjournals.org/content/early/2011/06/06/nar.gkr439.full
Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia.
Xose S. Puente, Magda Pinyol, Víctor Quesada, Laura Conde, Gonzalo R. Ordóñez, Neus Villamor, Georgia Escaramis, Pedro Jares, Sílvia Beà, Marcos González-Díaz, Laia Bassaganyas, Tycho Baumann, Manel Juan, Mónica López-Guerra, Dolors Colomer, José M. C. Tubío, Cristina López, Alba Navarro, Cristian Tornador, Marta Aymerich, María Rozman, Jesús M. Hernández, Diana A. Puente, José M. P. Freije, Gloria Velasco, Ana Gutiérrez-Fernández, Dolors Costa, Anna Carrió, Sara Guijarro, Anna Enjuanes, Lluís Hernández, Jordi Yagüe, Pilar Nicolás, Carlos M. Romeo-Casabona, Heinz Himmelbauer, Ester Castillo, Juliane C. Dohm, Silvia de Sanjosé, Miguel A. Piris, Enrique de Alava, Jesús San Miguel, Romina Royo, Josep L. Gelpí, David Torrents, Modesto Orozco,David G Pisano, Alfonso Valencia, Roderic Guigó, Mónica Bayés, Simon Heath, Marta Gut, Peter Klatt, John Marshall, Keiran Raine, Lucy A. Stebbings, P. Andrew Futreal, Michael R. Stratton, Peter J. Campbell, Ivo Gut, Armando López-Guillermo, Xavier Estivill, Emili Montserrat, Carlos López-Otín and Elías Campo.
Nature. Published on line 5th June, 2011.
Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.
Further info: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10113.html
MicroRNA expression in diffuse large B-cell lymphoma treated with chemoimmunotherapy.
Montes-Moreno S, Martinez N, Sanchez-Espiridión B, Díaz Uriarte R, Rodriguez ME, Saez A, Montalbán C, Gómez G, Pisano DG, García JF, Conde E, Gonzalez-Barca E, Lopez A, Mollejo M, Grande C, Martinez MA, Dunphy C, Hsi ED, Rocque GB, Chang J, Go RS, Visco C, Xu-Monette Z, Young KH, Piris MA.
Blood 2011 Jul 28;118(4):1034-40.
DLBCL prognostication requires additional biological markers. MicroRNAs (miRNAs) may constitute markers for cancer diagnosis, outcome or therapy response. Here we have analyzed the miRNA expression profile in a retrospective multicenter series of DLBCL patients (258 cases) uniformly treated with chemoimmunotherapy. Findings were correlated with OS and PFS. miRNA and gene expression profiles were studied using microarrays in an initial set of 36 cases. A selection of miRNAs associated with either DLBCL molecular subtypes (GCB/ABC) or clinical outcome were studied by multiplex RT-PCR in a test group of 240 cases with available FFPE diagnostic sample, divided into a training set (123 patients), which was used to derive miRNA-based and combined (with IPI score) Cox regression models, and an independent validation series (117 patients). A model based on miRNA expression predicts OS and PFS. This model improves the prediction based on clinical variables. Combined models with IPI score identify a high-risk group of patients with a 2-year OS and PFS probability of less than 50%. In summary, a precise miRNA signature is associated with poor clinical outcome in chemoimmunotherapy-treated DLBCL patients. This information improves IPI-based prediction and identifies a subgroup of candidate patients for alternative therapeutic regimens to those of standard therapies.
Further info: http://www.ncbi.nlm.nih.gov/pubmed/21633089
A DNA methylation fingerprint of 1,628 human samples.
Fernandez AF, Assenov Y, Martin-Subero J, Balint B, Siebert R, Taniguchi H, Yamamoto H, Hidalgo M, Tan AC, Galm O, Ferrer I, Sanchez-Cespedes M, Villanueva A, Carmona J, Sanchez-Mut JV, Berdasco M, Moreno V, Capella G, Monk D, Ballestar E, Ropero S, Martinez R, Sanchez-Carbayo M, Prosper F, Agirre X, Fraga MF, Graña O, Perez-Jurado L, Mora J, Puig S, Prat J, Badimon L, Puca AA, Meltzer SJ, Lengauer T, Bridgewater J, Bock C, Esteller M.
Genome Res. 2011 May 25.
DNA methylation is the best characterized of the different layers that make up the epigenetic setting. Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. The recently arrived single-base-resolution technologies for DNA methylation are extremely helpful tools, but are not yet applicable and affordable for studying large groups of subjects. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1,628 human samples where we interrogated 1,505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG island hypermethylation and a loss of CpG methylation in non-CpG island promoters. Although transformed cells are those where DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the obtained DNA methylation fingerprints might be useful for translational purposes by showing that are able to identify the tumor type origin of Cancers of Unknown Primary (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps, and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases.
Further info: http://genome.cshlp.org/content/early/2011/05/25/gr.119867.110.long
Epigenetic regulation of Nanog expression by Ezh2 in pluripotent stem cells.
Villasante A, Piazzolla D, Li H, Gómez-López G, Djabali M, Serrano M.
Cell Cycle 2011 May 1;10(9).
Nanog levels in pluripotent stem cells are heterogeneous and this is thought to reflect two different and interchangeable cell states, respectively poised to self-renew (Nanog-high subpopulation) or to differentiate (Nanog-low subpopulation). However, little is known about the mechanisms responsible for this pattern of Nanog expression. Here, we have examined the impact of the histone methyltransferase Ezh2 on pluripotent stem cells and on Nanog expression. Interestingly, induced pluripotent stem (iPS) cells lacking Ezh2 presented higher levels of Nanog due to a relative expansion of the Nanog-high subpopulation, and this was associated to severe defects in differentiation. Moreover, we found that the Nanog promoter in embryonic stem (ES) cells and iPS cells coexists in two alternative univalent chromatin configurations, either H3K4me3 or H3K27me3, the latter being dependent on the presence of functional Ezh2. Finally, the levels of expression of Ezh2, as well as the amount of H3K27me3 present at the Nanog promoter, were higher in the Nanog-low subpopulation of ES/iPS cells. Together, these data indicate that Ezh2 directly regulates the epigenetic status of the Nanog promoter affecting the balance of Nanog expression in pluripotent stem cells and, therefore, the equilibrium between self-renewal and differentiation.
Further info: http://www.landesbioscience.com/journals/cc/article/15658/
Combinatorial effects of microRNAs to suppress the Myc oncogenic pathway.
Bueno MJ, Gómez de Cedrón M, Gómez-López G, Perez de Castro I, Di Lisio L, Montes S, Martinez N, Guerrero M, Sanchez-Martinez R, Santos J, Pisano DG, Piris MA, Fernández-Piqueras J, Malumbres M.
Blood 2011, Apr 8.
Many mammalian transcripts contain target sites for multiple miRNAs although it is not clear to what extent miRNAs may coordinately regulate single genes. We have mapped the interactions between downregulated miRNAs and overexpressed target protein-coding genes in murine and human lymphomas. Myc, one of the hallmark oncogenes in these lymphomas, stands out as the upregulated gene with the highest number of genetic interactions with downregulated miRNAs in mouse lymphomas. The regulation of Myc by several of these miRNAs is confirmed by cellular and reporter assays. The same approach indentifies MYC and multiple Myc targets as a preferential target of downregulated miRNAs in human Burkitt's lymphoma, a pathology characterized by translocated MYC oncogenes. These results indicate that several miRNAs must be coordinately downregulated in order to enhance critical oncogenes such as Myc. Some of these Myc-targeting miRNAs are repressed by Myc, suggesting that these tumors are a consequence of the unbalanced activity of Myc versus miRNAs.
Further info: http://bloodjournal.hematologylibrary.org/content/early/2011/04/08/blood-2010-10-315432.abstract
Assessment of copy number variation using the Illumina Infinium 1M SNP-array: a comparison of methodological approaches in the Spanish Bladder Cancer/EPICURO study.
Gaëlle Marenne, Benjamín Rodríguez-Santiago, Montserrat García Closas, Luis Pérez-Jurado, Nathaniel Rothman, Daniel Rico, Guillermo Pita, David G. Pisano, Manolis Kogevinas, Debra T. Silverman, Alfonso Valencia, Francisco X. Real, Stephen J. Chanock, Emmanuelle Génin, Núria Malats. Hum Mutat (2011) vol. 32 (2) pp. 240-8. High-throughput single nucleotide polymorphism (SNP)-array technologies allow to investigate copy number variants (CNVs) in genome-wide scans and specific calling algorithms have been developed to determine CNV location and copy number. We report the results of a reliability analysis comparing data from 96 pairs of samples processed with CNVpartition, PennCNV, and QuantiSNP for Infinium Illumina Human 1Million probe chip data. We also performed a validity assessment with multiplex ligation-dependent probe amplification (MLPA) as a reference standard. The number of CNVs per individual varied according to the calling algorithm. Higher numbers of CNVs were detected in saliva than in blood DNA samples regardless of the algorithm used. All algorithms presented low agreement with mean Kappa Index (KI) 0.62). Our results indicate that the current calling algorithms should be improved for high performance CNV analysis in genome-wide scans.
PileLine: a toolbox to handle genome position information in next-generation sequencing studies
Daniel Glez-Peña , Gonzalo Gomez-Lopez , Miguel Reboiro-Jato , Florentino Fdez-Riverola and David G. Pisano
BMC Bioinformatics 2011, Jan 24;12(1):31. Highly accessed article.
Genomic position (GP) files currently used in next-generation sequencing (NGS) studies are always difficult to manipulate due to their huge size and the lack of appropriate tools to properly manage them. The structure of these flat files is based on representing one line per position that has been covered by at least one aligned read, imposing significant restrictions from a computational performance perspective.
PileLine implements a flexible command-line toolkit providing specific support to the management, filtering, comparison and annotation of GP files produced by NGS experiments. PileLine tools are coded in Java and run on both UNIX (Linux, Mac OS) and Windows platforms. The set of tools comprising PileLine are designed to be memory efficient by performing fast seek on-disk operations over sorted GP files.
Our novel toolbox has been extensively tested taking into consideration performance issues. It is publicly available at http://sourceforge.net/projects/pilelinetools under the GNU LGPL license. Full documentation including common use cases and guided analysis workflows is available at http://sing.ei.uvigo.es/pileline.
Further info: www.biomedcentral.com/1471-2105/12/31
Expression and functional validation of new p38α transcriptional targets in tumorigenesis.
Swat A, Dolado I, Igea A, Gómez-López G, Pisano DG, Cuadrado A, Nebreda AR.
Biochem J. Feb 24;434(3):549-58. 2011. p38α MAP kinase plays an important tumor suppressor role, which is mediated by both its negative effect on cell proliferation and its pro-apoptotic activity. Surprisingly, most tumor suppressor mechanisms coordinated by p38α have been reported to occur at the post- translational level. This contrasts with the important role of p38α in the regulation of transcription and the profound changes in gene expression that normally occur during tumorigenesis. We have analyzed whole genome expression profiles of Ras-transformed wild- type and p38α-deficient cells and have identified 202 genes that are potentially regulated by p38α in transformed cells. Expression analysis has confirmed the regulation of these genes by p38α in tumors, and functional validation has identified several of them as likely mediators of the tumor suppressor effect of p38α on Ras-induced transformation. Interestingly, about 10% of the genes that are negatively regulated by p38α in transformed cells contribute to EGF receptor signalling. Our results suggest that inhibition of EGF receptor signalling by transcriptional targets of p38α is an important function of this signalling pathway in the context of tumor suppression.
Further info: http://www.biochemj.org/bj/imps/abs/BJ20101410.htm#
Massive parallel DNA pyrosequencing analysis of the tumor suppressor BRG1/SMARCA4 in lung primary tumors.
Rodríguez-Nieto S, Cañada A, Pros E, Pinto AI, Torres-Lanzas J, Lopez-Rios F, Sánchez-Verde L, Pisano DG, Sánchez-Cespedes M.
Hum Mutat. 2010 Dec 7.The tumor suppressor gene, SMARCA4 (or BRG1), which encodes the ATPase component of the chromatin remodeling complex SWI/SNF, is commonly inactivated by mutations and deletions in lung cancer cell lines. However, SMARCA4 alterations appear to be rare in lung primary tumors. Ultra-deep sequencing technologies provide a promising alternative to achieve a sensitivity superior to that of current sequencing strategies. Here we used ultra-deep pyrosequencing to screen for mutations over the entire SMARCA4 coding region in 12 lung tumors without detectable BRG1 protein. While automatic-fluorescence-based sequencing detected one somatic mutation (p.K586X), the pyrosequencing revealed additional variants, thus increasing the sensitivity. One of the variants, which affected a consensus splice site, was confirmed by individual cloning of PCR products, ruling out the possibility of PCR or pyrosequencing artifacts. This mutation, confirmed to be somatic, was present at a frequency of ten percent, suggesting normal cell contamination in the tumor. Our analysis also allowed us to determine the sensitivity and to identify some limitations of the technology. In conclusion, in addition to cell lines, SMARCA4 is biallelically inactivated in a significant proportion of lung primary tumors, thereby constituting one of the most important genes contributing to the development of this type of cancer.
Further info: http://onlinelibrary.wiley.com/doi/10.1002/humu.21415/abstract;jsessionid=A8C042A523D2E07303479A7AFA4A6768.d02t01
Transcriptional profiling reveals different pseudo-hypoxic signatures in SDHB and VHL-related pheocromocytomas.
Elena López-Jiménez, Gonzalo Gómez-López, L Javier Leandro-García, Iván Muñoz, Francesca Schiavi, Cristina Montero-Conde, Aguirre A de Cubas, Ricardo Ramires, Iñigo Landa, Susanna Leskelä, Agnieszka Maliszewska, Lucía Inglada-Pérez, Leticia de la Vega, Cristina Rodríguez-Antona, Rocío Letón, Carmen Bernal, José M de Campos, Cristina Diez-Tascón, Mario F Fraga, Cesar Boullosa, David G Pisano, Giuseppe Opocher, Mercedes Robledo, Alberto Cascón.
Mol. Endocrinol. (2010) Dec;24(12):2382-91.
Further info: http://www.biomedcentral.com/1755-8794/3/44
Transcriptional characteristics of familial non-BRCA1/BRCA2 breast tumors.
Ricardo Fernández-Ramires, Gonzalo Gómez, Iván Muñoz-Repeto, Loris de Cecco, Gemma Llort, Alicia Cazorla, Ignacio Blanco, Manuela Gariboldi, Marco Alessandro Pierotti, Javier Benítez, Ana Osorio.
Int J Cancer (2010)In order to better understand the alterations present in the group of the so- called BRCAX tumors, we have used a cDNA microarray containing genes related to tumorigenesis and analyzed a series of 49 tumors consisting of 13 BRCA1, 14 BRCAX and 22 sporadic. We have confirmed that the BRCAX tumors are heterogeneous and can be divided in at least two main subgroups, so-called A and B, transcriptionally distinguishable and with different altered pathways within each of the groups. We have found that BRCAX-A and B subgroups, can be classified as Luminal A and B respectively, taking into account the intrinsic phenotypes defined for the sporadic breast tumors. We have found that, at the somatic level, the BRCAX-B tumors are identical to their sporadic Luminal B counterparts, while BRCAX-A, despite having a luminal A phenotype, show additional genomic alterations. We have found twenty-one de-regulated genes in the BRCAX-A group that we have called "The BRCAX Susceptibility Pathway" and suggested it as a candidate to search for new genes involved in the inherited susceptibility underlying the disease in this group.
Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.
Paula Martinez, Maria Thanasoula, Ana R Carlos, Gonzalo Gómez-López, Agueda M Tejera, Stefan Schoeftner, Orlando Dominguez, David G Pisano, Madalena Tarsounas, Maria A Blasco.
Nature Cell Biology (2010) vol. 12 (8) pp. 768-80
Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)(2) consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation.
Further info: http://www.nature.com/ncb/journal/v12/n8/abs/ncb2081.html
waviCGH: a web application for the analysis and visualization of genomic copy number alterations.
Angel Carro, Daniel Rico, Oscar M Rueda, Ramón Díaz-Uriarte, David G Pisano.
Nucleic Acids Res (2010) vol. 38 Suppl pp. W182-7
waviCGH is a versatile web server for the analysis and comparison of genomic copy number alterations in multiple samples from any species. waviCGH processes data generated by high density SNP-arrays, array-CGH or copy-number calls generated by any technique. waviCGH includes methods for pre-processing of the data, segmentation, calling of gains and losses, and minimal common regions determination over a set of experiments. The server is a user-friendly interface to the analytical methods, with emphasis on results visualization in a genomic context. Analysis tools are introduced to the user as the different steps to follow in an experimental protocol. All the analysis steps generate high quality images and tables ready to be imported into spreadsheet programs. Additionally, for human, mouse and rat, altered regions are represented in a biological context by mapping them into chromosomes in an integrated cytogenetic browser. waviCGH is available at http://wavi.bioinfo.cnio.es.
Further info: http://nar.oxfordjournals.org/cgi/content/full/38/suppl_2/W182?view=long&pmid=20507915
The EMBRACE web service collection.
Steve Pettifer, Jon Ison, Matús Kalas, Dave Thorne, Philip McDermott, Inge Jonassen, Ali Liaquat, José M Fernández, Jose M Rodriguez, INB-Partners, David G Pisano, Christophe Blanchet, Mahmut Uludag, Peter Rice, Edita Bartaseviciute, Kristoffer Rapacki, Maarten Hekkelman, Olivier Sand, Heinz Stockinger, Andrew B Clegg, Erik Bongcam-Rudloff, Jean Salzemann, Vincent Breton, Teresa K Attwood, Graham Cameron, Gert Vriend.
Nucleic Acids Res (2010) vol. 38 Suppl pp. W683-8
The EMBRACE (European Model for Bioinformatics Research and Community Education) web service collection is the culmination of a 5-year project that set out to investigate issues involved in developing and deploying web services for use in the life sciences. The project concluded that in order for web services to achieve widespread adoption, standards must be defined for the choice of web service technology, for semantically annotating both service function and the data exchanged, and a mechanism for discovering services must be provided. Building on this, the project developed: EDAM, an ontology for describing life science web services; BioXSD, a schema for exchanging data between services; and a centralized registry (http://www.embraceregistry.net) that collects together around 1000 services developed by the consortium partners. This article presents the current status of the collection and its associated recommendations and standards definitions.
Further info: http://nar.oxfordjournals.org/cgi/content/full/38/suppl_2/W683?view=long&pmid=20462862
Mantle cell lymphoma: transcriptional regulation by microRNAs.
L Di Lisio, G Gómez-López, M Sánchez-Beato, C Gómez-Abad, M E Rodríguez, R Villuendas, B I Ferreira, Carro, D Rico, M Mollejo, M A Martínez, J Menárguez, A Díaz-Alderete, J Gil, J C Cigudosa, David G Pisano, M A Piris, N Martínez.
Leukemia (2010) vol. 24 (7) pp. 1335-42
Mantle cell lymphoma (MCL) pathogenesis is still partially unexplained. We investigate the importance of microRNA (miRNA) expression as an additional feature that influences MCL pathway deregulation and may be useful for predicting patient outcome. Twenty-three MCL samples, eight cell lines and appropriate controls were screened for their miRNAs and gene expression profiles and DNA copy-number changes. MCL patients exhibit a characteristic signature that includes 117 miRNA (false discovery rate <0.05). Combined analysis of miRNAs and the gene expression profile, paired with bioinformatics target prediction (miRBase and TargetScan), revealed a series of genes and pathways potentially targeted by a small number of miRNAs, including essential pathways for lymphoma survival such as CD40, mitogen-activated protein kinase and NF-kappaB. Functional validation in MCL cell lines demonstrated NF-kappaB subunit nuclear translocation to be regulated by the expression of miR-26a. The expression of 12 selected miRNAs was studied by quantitative PCR in an additional series of 54 MCL cases. Univariate analysis identified a single miRNA, miR-20b, whose lack of expression distinguished cases with a survival probability of 56% at 60 months. In summary, using a novel bioinformatics approach, this study identified miRNA changes that contribute to MCL pathogenesis and markers of potential utility in MCL diagnosis and clinical prognostication.
Further info: http://www.nature.com/leu/journal/v24/n7/abs/leu201091a.html
MidA is a putative methyltransferase that is required for mitochondrial complex I function.
Sergio Carilla-Latorre, M Esther Gallardo, Sarah J Annesley, Javier Calvo-Garrido, Osvaldo Graña, Sandra L Accari, Paige K Smith, Alfonso Valencia, Rafael Garesse, Paul R Fisher, Ricardo Escalante.
J Cell Sci (2010) vol. 123 (Pt 10) pp. 1674-83
Dictyostelium and human MidA are homologous proteins that belong to a family of proteins of unknown function called DUF185. Using yeast two-hybrid screening and pull-down experiments, we showed that both proteins interact with the mitochondrial complex I subunit NDUFS2. Consistent with this, Dictyostelium cells lacking MidA showed a specific defect in complex I activity, and knockdown of human MidA in HEK293T cells resulted in reduced levels of assembled complex I. These results indicate a role for MidA in complex I assembly or stability. A structural bioinformatics analysis suggested the presence of a methyltransferase domain; this was further supported by site-directed mutagenesis of specific residues from the putative catalytic site. Interestingly, this complex I deficiency in a Dictyostelium midA(-) mutant causes a complex phenotypic outcome, which includes phototaxis and thermotaxis defects. We found that these aspects of the phenotype are mediated by a chronic activation of AMPK, revealing a possible role of AMPK signaling in complex I cytopathology.
Further info: http://jcs.biologists.org/cgi/content/full/123/10/1674
Serum and tissue profiling in bladder cancer combining protein and tissue arrays.
Esteban Orenes-Piñero, Rodrigo Barderas, Daniel Rico, J Ignacio Casal, David G Pisano, Jose Navajo, Ferran Algaba, Josep Maria Piulats, Marta Sanchez-Carbayo.
J Proteome Res (2010) vol. 9 (1) pp. 164-73
Aiming at identifying biomarkers for bladder cancer, the serum proteome was explored in a pilot study through a profiling approach using protein arrays. Supervised analyses identified a panel 171 immunogenic proteins differentially expressed between patients with bladder cancer (n = 12) and controls without the disease (n = 10). The microanatomical expression patterns of novel immunogenic proteins, especially dynamin and clusterin, were found significantly associated with histopathologic variables and overall survival, as confirmed by immunohistochemistry using an independent series of bladder tumors contained in tissue microarrays (n = 289). Thus, the protein arrays approach has identified a panel of immunogenic candidates that may potentially play a role as diagnostic biomarkers, especially for muscle invasive disease. Moreover, the protein expression patterns of dynamin and clusterin in bladder tumors were shown to adjunct for histopathologic staging and clinical outcome prognosis.
Further info: http://pubs.acs.org/doi/abs/10.1021/pr900273u
Inference of functional relations in predicted protein networks with a machine learning approach.
Beatriz García-Jiménez, David Juan, Iakes Ezkurdia, Eduardo Andrés-León, Alfonso Valencia.
PLoS ONE (2010) vol. 5 (4).
BACKGROUND: Molecular biology is currently facing the challenging task of functionally characterizing the proteome. The large number of possible protein-protein interactions and complexes, the variety of environmental conditions and cellular states in which these interactions can be reorganized, and the multiple ways in which a protein can influence the function of others, requires the development of experimental and computational approaches to analyze and predict functional associations between proteins as part of their activity in the interactome. METHODOLOGY/PRINCIPAL FINDINGS: We have studied the possibility of constructing a classifier in order to combine the output of the several protein interaction prediction methods. The AODE (Averaged One-Dependence Estimators) machine learning algorithm is a suitable choice in this case and it provides better results than the individual prediction methods, and it has better performances than other tested alternative methods in this experimental set up. To illustrate the potential use of this new AODE-based Predictor of Protein InterActions (APPIA), when analyzing high-throughput experimental data, we show how it helps to filter the results of published High-Throughput proteomic studies, ranking in a significant way functionally related pairs. Availability: All the predictions of the individual methods and of the combined APPIA predictor, together with the used datasets of functional associations are available at http://ecid.bioinfo.cnio.es/. CONCLUSIONS: We propose a strategy that integrates the main current computational techniques used to predict functional associations into a unified classifier system, specifically focusing on the evaluation of poorly characterized protein pairs. We selected the AODE classifier as the appropriate tool to perform this task. AODE is particularly useful to extract valuable information from large unbalanced and heterogeneous data sets. The combination of the information provided by five prediction interaction prediction methods with some simple sequence features in APPIA is useful in establishing reliability values and helpful to prioritize functional interactions that can be further experimentally characterized.
Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=20376314&retmode=ref&cmd=prlinks
Whole genome analysis of p38 SAPK-mediated gene expression upon stress.
Isabel Ferreiro, Manel Joaquin, Abul Islam, Gonzalo Gomez-Lopez, Montserrat Barragan, Luís Lombardía, Orlando Domínguez, David G Pisano, Nuria Lopez-Bigas, Angel R Nebreda, Francesc Posas. BMC Genomics (2010) vol. 11.
BACKGROUND: Cells have the ability to respond and adapt to environmental changes through activation of stress-activated protein kinases (SAPKs). Although p38 SAPK signalling is known to participate in the regulation of gene expression little is known on the molecular mechanisms used by this SAPK to regulate stress-responsive genes and the overall set of genes regulated by p38 in response to different stimuli. RESULTS: Here, we report a whole genome expression analyses on mouse embryonic fibroblasts (MEFs) treated with three different p38 SAPK activating-stimuli, namely osmostress, the cytokine TNFalpha and the protein synthesis inhibitor anisomycin. We have found that the activation kinetics of p38alpha SAPK in response to these insults is different and also leads to a complex gene pattern response specific for a given stress with a restricted set of overlapping genes. In addition, we have analysed the contribution of p38alpha the major p38 family member present in MEFs, to the overall stress-induced transcriptional response by using both a chemical inhibitor (SB203580) and p38alpha deficient (p38alpha-/-) MEFs. We show here that p38 SAPK dependency ranged between 60% and 88% depending on the treatments and that there is a very good overlap between the inhibitor treatment and the ko cells. Furthermore, we have found that the dependency of SAPK varies depending on the time the cells are subjected to osmostress. CONCLUSIONS: Our genome-wide transcriptional analyses shows a selective response to specific stimuli and a restricted common response of up to 20% of the stress up-regulated early genes that involves an important set of transcription factors, which might be critical for either cell adaptation or preparation for continuous extra-cellular changes. Interestingly, up to 85% of the up-regulated genes are under the transcriptional control of p38 SAPK. Thus, activation of p38 SAPK is critical to elicit the early gene expression program required for cell adaptation to stress.
Further info: http://www.biomedcentral.com/1471-2164/11/144
Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations.
Stefan Schoeftner, Raquel Blanco, Isabel Lopez de Silanes, Purificación Muñoz, Gonzalo Gómez-López, Juana M Flores, Maria A Blasco.
Proc Natl Acad Sci USA (2009) vol. 106 (46) pp. 19393-8.
Telomeres are heterochromatic structures at chromosome ends essential for chromosomal stability. Telomere shortening and the accumulation of dysfunctional telomeres are associated with organismal aging. Using telomerase-deficient TRF2-overexpressing mice (K5TRF2/Terc(-/-)) as a model for accelerated aging, we show that telomere shortening is paralleled by a gradual deregulation of the mammalian transcriptome leading to cumulative changes in a defined set of genes, including up-regulation of the mTOR and Akt survival pathways and down-regulation of cell cycle and DNA repair pathways. Increased DNA damage from dysfunctional telomeres leads to reduced deposition of H3K27me3 onto the inactive X chromosome (Xi), impaired association of the Xi with telomeric transcript accumulations (Tacs), and reactivation of an X chromosome-linked K5TRF2 transgene that is subjected to X-chromosome inactivation in female mice with sufficiently long telomeres. Exogenously induced DNA damage also disrupts Xi-Tacs, suggesting DNA damage at the origin of these alterations. Collectively, these findings suggest that critically short telomeres activate a persistent DNA damage response that alters gene expression programs in a nonstochastic manner toward cell cycle arrest and activation of survival pathways, as well as impacts the maintenance of epigenetic memory and nuclear organization, thereby contributing to organismal aging.
Further info: http://www.pnas.org/content/106/46/19393.long
Assessment of domain boundary predictions and the prediction of intramolecular contacts in CASP8.
Ezkurdia I, Graña O, Izarzugaza JM, Tress ML.
Proteins. 2009;77 Suppl 9:196-209.
This article details the assessment process and evaluation results for two categories in the 8th Critical Assessment of Protein Structure Prediction experiment (CASP8). The domain prediction category was evaluated with a range of scores including the Normalized Domain Overlap score and a domain boundary distance measure. Residue-residue contact predictions were evaluated with standard CASP measures, prediction accuracy, and Xd. In the domain boundary prediction category, prediction methods still make reliable predictions for targets that have structural templates, but continue to struggle to make good predictions for the few ab initio targets in CASP. There was little indication of improvement in the domain prediction category. The contact prediction category demonstrated that there was renewed interest among predictors and despite the small sample size the results suggested that there had been an increase in prediction accuracy. In contrast to CASP7 contact specialists predicted contacts more accurately than the majority of tertiary structure predictors. Despite this small success, the lack of free modeling targets makes it unlikely that either category will be included in their present form in CASP9.
Functional signatures identified in B-cell non-Hodgkin lymphoma profiles.
Mohit Aggarwal, Margarita Sánchez-Beato, Gonzalo Gómez-López, Fátima Al-Shahrour, Nerea Martínez, Antonia Rodríguez, Elena Ruiz-Ballesteros, Francisca I Camacho, Alberto Pérez-Rosado, Paloma de la Cueva, María J Artiga, David G Pisano, Eva Kimby, Joaquín Dopazo, Raquel Villuendas, Miguel A Piris.
Leuk Lymphoma (2009) vol. 50 (10) pp. 1699-708
Gene-expression profiling in B-cell lymphomas has provided crucial data on specific lymphoma types, which can contribute to the identification of essential lymphoma survival genes and pathways. In this study, the gene-expression profiling data of all major B-cell lymphoma types were analyzed by unsupervised clustering. The transcriptome classification so obtained, was explored using gene set enrichment analysis generating a heatmap for B-cell lymphoma that identifies common lymphoma survival mechanisms and potential therapeutic targets, recognizing sets of coregulated genes and functional pathways expressed in different lymphoma types. Some of the most relevant signatures (stroma, cell cycle, B-cell receptor (BCR)) are shared by multiple lymphoma types or subclasses. A specific attention was paid to the analysis of BCR and coregulated pathways, defining molecular heterogeneity within multiple B-cell lymphoma types.
Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=19863341&retmode=ref&cmd=prlinks
WhichGenes: a web-based tool for gathering, building, storing and exporting gene sets with application in gene set enrichment analysis.
Daniel Glez-Peña, Gonzalo Gómez-López, David G Pisano, Florentino Fdez-Riverola.
Nucleic Acids Res (2009) vol. 37 (Web Server issue) pp. W329-34
WhichGenes is a web-based interactive gene set building tool offering a very simple interface to extract always-updated gene lists from multiple databases and unstructured biological data sources. While the user can specify new gene sets of interest by following a simple four-step wizard, the tool is able to run several queries in parallel. Every time a new set is generated, it is automatically added to the private gene-set cart and the user is notified by an e-mail containing a direct link to the new set stored in the server. WhichGenes provides functionalities to edit, delete and rename existing sets as well as the capability of generating new ones by combining previous existing sets (intersection, union and difference operators). The user can export his sets configuring the output format and selecting among multiple gene identifiers. In addition to the user-friendly environment, WhichGenes allows programmers to access its functionalities in a programmatic way through a Representational State Transfer web service. WhichGenes front-end is freely available at http://www.whichgenes.org/, WhichGenes API is accessible at http://www.whichgenes.org/api/.
Further info: http://nar.oxfordjournals.org/cgi/content/full/37/suppl_2/W329?view=long&pmid=19406925
The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer.
Agustin F Fernandez, Cecilia Rosales, Pilar Lopez-Nieva, Osvaldo Graña, Esteban Ballestar, Santiago Ropero, Jesus Espada, Sonia A Melo, Amaia Lujambio, Mario F Fraga, Irene Pino, Biola Javierre, Francisco J Carmona, Francesco Acquadro, Renske D M Steenbergen, Peter J F Snijders, Chris J Meijer, Pascal Pineau, Anne Dejean, Belen Lloveras, Gabriel Capella, Josep Quer, Maria Buti, Juan-Ignacio Esteban, Helena Allende, Francisco Rodriguez-Frias, Xavier Castellsague, Janos Minarovits, Jordi Ponce, Daniela Capello, Gianluca Gaidano, Juan Cruz Cigudosa, Gonzalo Gomez-Lopez, David G Pisano, Alfonso Valencia, Miguel Angel Piris, Francesc X Bosch, Ellen Cahir-McFarland, Elliott Kieff, Manel Esteller.
Genome Res (2009) vol. 19 (3) pp. 438-51
The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses.
Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=19208682&retmode=ref&cmd=prlinks
Functional characterization of E- and P-cadherin in invasive breast cancer cells.
David Sarrió, José Palacios, Marta Hergueta-Redondo, Gonzalo Gómez-López, Amparo Cano, Gema Moreno-Bueno.
BMC Cancer (2009) vol. 9.
BACKGROUND: Alterations in the cadherin-catenin adhesion complexes are involved in tumor initiation, progression and metastasis. However, the functional implication of distinct cadherin types in breast cancer biology is still poorly understood. METHODS: To compare the functional role of E-cadherin and P-cadherin in invasive breast cancer, we stably transfected these molecules into the MDA-MB-231 cell line, and investigated their effects on motility, invasion and gene expression regulation. RESULTS: Expression of either E- and P-cadherin significantly increased cell aggregation and induced a switch from fibroblastic to epithelial morphology. Although expression of these cadherins did not completely reverse the mesenchymal phenotype of MDA-MB-231 cells, both E- and P-cadherin decreased fibroblast-like migration and invasion through extracellular matrix in a similar way. Moreover, microarray gene expression analysis of MDA-MB-231 cells after expression of E- and P-cadherins revealed that these molecules can activate signaling pathways leading to significant changes in gene expression. Although the expression patterns induced by E- and P-cadherin showed more similarities than differences, 40 genes were differentially modified by the expression of either cadherin type. CONCLUSION: E- and P-cadherin have similar functional consequences on the phenotype and invasive behavior of MDA-MB-231 cells. Moreover, we demonstrate for the first time that these cadherins can induce both common and specific gene expression programs on invasive breast cancer cells. Importantly, these identified genes are potential targets for future studies on the functional consequences of altered cadherin expression in human breast cancer.
Further info: http://www.biomedcentral.com/1471-2407/9/74
Translational disease interpretation with molecular networks.
Anaïs Baudot, Gonzalo Gómez-López, Alfonso Valencia.
Genome Biol (2009) vol. 10 (6) pp. 221
Molecular networks are being used to reconcile genotypes and phenotypes by integrating medical information. In this context, networks will be instrumental for the interpretation of disease at the personalized medicine level.
Further info: http://genomebiology.com/content/10/6/221
EcID: A database for the inference of functional interactions in E. coli.
Eduardo Andres Leon, Iakes Ezkurdia, Beatriz García, Alfonso Valencia, David Juan.
Nucleic Acids Res (2009) vol. 37 (Database issue) pp. D629-35
The EcID database (Escherichia coli Interaction Database) provides a framework for the integration of information on functional interactions extracted from the following sources: EcoCyc (metabolic pathways, protein complexes and regulatory information), KEGG (metabolic pathways), MINT and IntAct (protein interactions). It also includes information on protein complexes from the two E. coli high-throughput pull-down experiments and potential interactions extracted from the literature using the web services associated to the iHOP text-mining system. Additionally, EcID incorporates results of various prediction methods, including two protein interaction prediction methods based on genomic information (Phylogenetic Profiles and Gene Neighbourhoods) and three methods based on the analysis of co-evolution (Mirror Tree, In Silico 2 Hybrid and Context Mirror). EcID associates to each prediction a specifically developed confidence score. The two main features that make EcID different from other systems are the combination of co-evolution-based predictions with the experimental data, and the introduction of E. coli-specific information, such as gene regulation information from EcoCyc. The possibilities offered by the combination of the EcID database information are illustrated with a prediction of potential functions for a group of poorly characterized genes related to yeaG. EcID is available online at http://ecid.bioinfo.cnio.es.
Further info: http://nar.oxfordjournals.org/cgi/content/full/37/suppl_1/D629
DAX1, a direct target of EWS/FLI1 oncoprotein, is a principal regulator of cell-cycle progression in Ewing's tumor cells.
E García-Aragoncillo, J Carrillo, E Lalli, N Agra, G Gómez-López, A Pestaña, J Alonso.
Oncogene (2008) vol. 27 (46) pp. 6034-43
The molecular hallmark of the Ewing's family of tumors is the presence of balanced chromosomal translocations, leading to the formation of chimerical transcription factors (that is, EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing's tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor that was not previously implicated in cancer, is induced by the EWS/FLI1 oncoprotein and is highly expressed in Ewing's tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this study we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGAA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing's cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about 10% of the genes regulated by EWS/FLI1 in Ewing's cells are DAX1 targets, confirming the importance of DAX1 in Ewing's oncogenesis. Functional genomic analysis, validated by quantitative RT-PCR, showed that genes implicated in cell-cycle progression, such as CDK2, CDC6, MCM10 or SKP2 were similarly regulated by EWS/FLI1 and DAX1. These findings indicate that DAX1 is important in the pathogenesis of the Ewing's family of tumors, identify new functions for DAX1 as a cell-cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference.
Further info: http://www.nature.com/onc/journal/v27/n46/full/onc2008203a.html
TCL1A expression delineates biological and clinical variability in B-cell lymphoma.
M Aggarwal, R Villuendas, G Gomez-Lopez, S Rodriguez-Pinilla, M Sanchez-Beato, D Alvarez, N Martinez, A Rodriguez, M Castillo, F Camacho, S Montes-Moreno, J Garcia-Marco, E Kimby, David G Pisano, MA Piris.
Mod Pathol (2008). Feb; 22(2):206-15.
The assembly of a collection of gene-expression signatures of the major types of B-cell non-Hodgkin's lymphoma has identified increased T-cell leukemia/lymphoma 1A (TCL1) expression in multiple lymphoma types and cases, and has enabled the investigation of the functional and clinical importance of TCL1 expression. Specifically, Burkitt's lymphoma cases show a homogeneously strong expression of TCL1, whereas diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, nodal marginal zone lymphoma, and splenic marginal zone lymphoma display a striking variability in the intensity of TCL1 staining. This was validated in two independent series. A Gene-Set Enrichment Analysis of the genes correlated with TCL1A expression found that variation in the level of expression of TCL1A was significantly associated with some of the most important gene signatures recognizing B-cell lymphoma pathogenesis and heterogeneity, such as germinal center, B-cell receptor, NF-kappaB (and its target genes), death, MAP kinases, TNFR1, TOLL, and IL1R. Additionally, TCL1 expression was correlated with shorter time to treatment in chronic lymphocytic leukemia cases and shorter lymphoma-specific survival in mantle cell lymphoma series, thus indicating the clinical and biological significance of TCL1 expression, and suggesting TCL1A as a potential therapeutic target.
Further info: http://www.ncbi.nlm.nih.gov/pubmed/18820675?dopt=abstract
miR-181b negatively regulates activation-induced cytidine deaminase in B cells.
V de Yébenes, Laura Belver, David G Pisano, S González, A Villasante, C Croce, L He, A Ramiro.
J Exp Med (2008). Sep 29; 205(10), 2199-206.
Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3' untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.
Further info: http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&id=18762567&retmode=ref&cmd=prlinks
Mechanistic principles of chromatin remodeling guided by siRNAs and miRNAs.
Susana Gonzalez, David G Pisano, Manuel Serrano.
Cell Cycle (2008) vol. 7 (16) pp. 2601-8
Small RNAs can guide chromatin remodeling in mammalian cells, but the mechanisms involved are poorly understood. Previous reports have shown a requirement for overlapping transcription and the involvement of Argonaute (Ago) proteins. Here, we use the Regulatory Domain (RD) of the INK4/ARF locus as an experimental platform susceptible to siRNA-guided chromatin remodeling to interrogate about the mechanisms involved. We show that siRNA-guided chromatin remodeling of RD requires overlapping transcription, and targets the transcribed strand, but not the template strand, supporting an RNA:RNA recognition mechanism between the small RNA and the nascent RNA transcript. We found that heterochromatin formation can be triggered both by perfectly-matched double-stranded RNA precursors, as well as, by imperfectly-matched double-stranded RNA precursors. These observations, together with the fact that promoters are often subjected to overlapping transcription, open the possibility that miRNAs could also be able to guide heterochromatin formation at promoters. We demonstrate this possibility showing that miRNAs miR17-5p and miR20a from the oncomiR cluster miR-17-92 can induce heterochromatic features in promoters that undergo overlapping transcription and possess sequence complementarity to the miRNA seed region. These results unveil a new level of gene regulation by miRNAs.
Further info: http://www.landesbioscience.com/journals/cc/article/6541/
Interoperability with Moby 1.0--it's better than sharing your toothbrush!.
BioMoby Consortium, Mark D Wilkinson, Martin Senger, Edward Kawas, Richard Bruskiewich, Jerome Gouzy, Celine Noirot, Philippe Bardou, Ambrose Ng, Dirk Haase, Enrique de Andres Saiz, Dennis Wang, Frank Gibbons, Paul M K Gordon, Christoph W Sensen, Jose Manuel Rodriguez Carrasco, José M Fernández, Lixin Shen, Matthew Links, Michael Ng, Nina Opushneva, Pieter B T Neerincx, Jack A M Leunissen, Rebecca Ernst, Simon Twigger, Bjorn Usadel, Benjamin Good, Yan Wong, Lincoln Stein, William Crosby, Johan Karlsson, Romina Royo, Iván Párraga, Sergio Ramírez, Josep Lluis Gelpi, Oswaldo Trelles, David G Pisano, Natalia Jimenez, Arnaud Kerhornou, Roman Rosset, Leire Zamacola, Joaquin Tarraga, Jaime Huerta-Cepas, Jose María Carazo, Joaquin Dopazo, Roderic Guigo, Arcadi Navarro, Modesto Orozco, Alfonso Valencia, M Gonzalo Claros, Antonio J Pérez, Jose Aldana-Montes, M Mar Rojano, Raul Fernandez-Santa Cruz, Ismael Navas, Gary Schiltz, Andrew Farmer, Damian Gessler, Heiko Schoof, Andreas Groscurth.
Brief Bioinformatics (2008) vol. 9 (3) pp. 220-31
The BioMoby project was initiated in 2001 from within the model organism database community. It aimed to standardize methodologies to facilitate information exchange and access to analytical resources, using a consensus driven approach. Six years later, the BioMoby development community is pleased to announce the release of the 1.0 version of the interoperability framework, registry Application Programming Interface and supporting Perl and Java code-bases. Together, these provide interoperable access to over 1400 bioinformatics resources worldwide through the BioMoby platform, and this number continues to grow. Here we highlight and discuss the features of BioMoby that make it distinct from other Semantic Web Service and interoperability initiatives, and that have been instrumental to its deployment and use by a wide community of bioinformatics service providers. The standard, client software, and supporting code libraries are all freely available at http://www.biomoby.org/.
Further info: http://bib.oxfordjournals.org/cgi/content/full/9/3/220
Bioinformatics and cancer research: building bridges for translational research.
Gonzalo Gómez-López, Alfonso Valencia.
Clinical & Translational Oncology. (2008) vol. 10 (2) pp. 85-95
Genome studies have revolutionised cancer research in recent years as high-throughput technologies can now be used to identify sets of genes potentially related with different processes in cancer. However, managing all this data and organising it into useful datasets is still a challenge in the bioinformatics field. Finding relationships between the molecular and genomic information and the clinical information available, within the medical informatics domain, is currently driving the development of translational research in biomedicine. The dispersion and complexity of the molecular information, the poor adherence to standards, together with the fast evolution of the experimental techniques, pose obvious challenges for the development of integrated molecular resources. In parallel, restricted access to medical information together with the gaps in the development of standard terminologies are typical limitations in the area of medical informatics. The development of research projects combining medical and molecular information together with the current efforts to standardise and integrate databases and terminologies are described in this review as a demonstration of the fruitful activity in this area.
Further info: http://www.ncbi.nlm.nih.gov/pubmed/18258507?dopt=abstract
Comparative genome profiling across subtypes of low-grade B-cell lymphoma identifies type-specific and common aberrations that target genes with a role in B-cell neoplasia.
Ferreira BI, García JF, Suela J, Mollejo M, Camacho FI, Carro A, Montes S, Piris MA, Cigudosa JC.
Haematologica. (2008) May; 93(5): 670-9.
BACKGROUND:Low-grade B-cell lymphomas are a very heterogeneous group of tumors, whose differential diagnosis is frequently compromised by the lack of specific cytogenetic or molecular features. Our objective was to search for genomic features that allow a better molecular identification of the different types of lymphoma studied. DESIGN AND METHODS:We selected a panel of 87 low-grade B-cell lymphoma tumor samples that were unambiguously diagnosed (clinically and cytogenetically) as: follicular, splenic marginal zone, nodal marginal zone, lymphoplasmacytic, mantle cell, extranodal marginal zone MALT-type lymphoma or B-cell chronic lymphocytic leukemia. All samples were subjected to the same high-resolution genomic DNA analysis (array-based comparative genomic hybridization): a whole genome platform that contained 44000 probes distributed across the genome. Genomic imbalances were recorded, compiled and analyzed. RESULTS:Eighty percent of analyzed cases showed genomic imbalances (deletions and gain/amplifications) but the frequency of these imbalances ranged from 100% in mantle cell lymphomas to 33% in MALT lymphomas. A total of 95 new genomic imbalances affecting all lymphoma subtypes, were defined. We evaluated the extension of the genomic instability, detecting distinct patterns of genomic instability within subtypes. Specific pathways, such as nuclear factor kB (gains of REL and BCL11A, and losses of COMMD3, BIRC1, IKK1 and NFKB2), Polycomb group proteins (gain of BMI1 and deletion of PCGF6), DNA repair checkpoint pathways (deletion of 16q24 involving CDT1), or miRNA with a role in B-cell lymphoma pathogenesis (MIRN15A, MIRN16-1), were targeted by this genomic instability. CONCLUSIONS: Although all subtypes of lymphomas showed gains and losses of DNA, the analysis of their genomic profiles indicated that there are specific aberrations in almost every subtype as well as frequent aberrations that are common to a large number of lymphoma types. These common aberrations target genes that are important in B-cell lymphomagenesis.
Further info: http://www.haematologica.org/cgi/content/full/93/5/670
CARGO: a web portal to integrate customized biological information.
Ildefonso Cases, David G Pisano, E Andres Leon, A. Carro, G Gomez-Lopez, JM Rodriguez, Jaime Fernández-Vera, Alfonso Valencia, Ana Rojas.
Nucleic Acids Res (2007). Jul 35 (Web Server issue):W16-20.
There is a huge quantity of information generated in Life Sciences, and it is dispersed in many databases and repositories. Despite the broad availability of the information, there is a great demand for methods that are able to look for, gather and display distributed data in a standardized and friendly way. CARGO (Cancer And Related Genes Online) is a configurable biological web portal designed as a tool to facilitate, integrate and visualize results from Internet resources, independently of their native format or access method. Through the use of small agents, called widgets, supported by a Rich Internet Application (RIA) paradigm based on AJAX, CARGO provides pieces of minimal, relevant and descriptive biological information. The tool is designed to be used by experimental biologists with no training in bioinformatics. In the current state, the system presents a list of human cancer genes. Available at http://cargo.bioinfo.cnio.es.
Further info: http://nar.oxfordjournals.org/cgi/content/abstract/gkm280v1