Background: Tobacco is the main risk factor for developing lung cancer. Yet, some heavy smokers do not develop lung cancer at advanced ages while others develop it at young ages. Here, we assess for the first time the genetic background of these clinically relevant extreme phenotypes using whole exome sequencing (WES). Methods: We performed WES of germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age ( extreme cases, n=50) or did not present lung adenocarcinoma or other tumors at an advanced age (extreme controls, n=50). We selected non-synonymous variants located in exonic regions and consensus splice sites of the genes that showed significantly different allelic frequencies between both cohorts. We validated our results in all the additional extreme cases (i.e., heavy smokers who developed lung adenocarcinoma at an early age) available from The Cancer Genome Atlas (TCGA). Results: The mean age for the extreme cases and controls was respectively 49.7 and 77.5 years. Mean tobacco consumption was 43.6 and 56.8 pack-years. We identified 619 significantly different variants between both cohorts, and we validated 108 of these in extreme cases selected from TCGA. Nine validated variants, located in relevant cancer related genes, such as PARP4, HLA-A or NQO1, among others, achieved statistical significance in the False Discovery Rate test. The most significant validated variant (P=4.48x10(-5)) was located in the tumor-suppressor gene ALPK2. Conclusions: We describe genetic variants associated with extreme phenotypes of high and low risk for the development of tobacco-induced lung adenocarcinoma. Our results and our strategy may help to identify high-risk subjects and to develop new therapeutic strategies.

Acute myeloid leukemia (AML) is an aggressive disease associated with very poor prognosis. Most patients are older than 60 years, and in this group only 5-15% of cases survive over 5 years. Therefore, it is urgent to develop more effective targeted therapies. Inactivation of protein phosphatase 2 A (PP2A) is a recurrent event in AML, and overexpression of its endogenous inhibitor SET is detected in similar to 30% of patients. The PP2A activating drug FTY720 has potent anti-leukemic effects; nevertheless, FTY720 induces cardiotoxicity at the anti-neoplastic dose. Here, we have developed a series of non-phosphorylable FTY720 analogues as a new therapeutic strategy for AML. Our results show that the lead compound CM-1231 re-activates PP2A by targeting SET-PP2A interaction, inhibiting cell proliferation and promoting apoptosis in AML cell lines and primary patient samples. Notably, CM-1231 did not induce cardiac toxicity, unlike FTY720, in zebrafish models, and reduced the invasion and aggressiveness of AML cells more than FTY720 in zebrafish xenograft models. In conclusion, CM-1231 is safer and more effective than FTY720; therefore, this compound could represent a novel and promising approach for treating AML patients with SET overexpression.

The protein detection and quantification using high-throughput proteomic technologies is still challenging due to the stochastic nature of the peptide selection in the mass spectrometer, the difficulties in the statistical analysis of the results and the presence of degenerated peptides. However, considering in the analysis only those peptides that could be detected by mass spectrometry, also called proteotypic peptides, increases the accuracy of the results. Several approaches have been applied to predict peptide detectability based on the physicochemical properties of the peptides. In this manuscript, we present DeepMSPeptide, a bioinformatic tool that uses a deep learning method to predict proteotypic peptides exclusively based on the peptide amino acid sequences.

The aim of this study was to characterize the effects of Maresin 1 (MaR1), a DHA-derived pro-resolving lipid mediator, on obesity-related colonic inflammation and gut dysbiosis in diet-induced obese (DIO) mice. In colonic mucosa of DIO mice, the MaR1 treatment decreased the expression of inflammatory genes, such asTnf-alpha andIl-1 beta. As expected, the DIO mice exhibited significant changes in gut microbiota composition at the phylum, genus, and species levels, with a trend to a higher Firmicutes/Bacteroidetes ratio. Deferribacteres and Synergistetes also increased in the DIO animals. In contrast, these animals exhibited a significant decrease in the content of Cyanobacteria and Actinobacteria. Treatment with MaR1 was not able to reverse the dysbiosis caused by obesity on the most abundant phyla. However, the MaR1 treatment increased the content ofP. xylanivorans, which have been considered to be a promising probiotic with healthy effects on gut inflammation. Finally, a positive association was found between the Deferribacteres andIl-1 beta expression, suggesting that the increase in Deferribacteres observed in obesity could contribute to the overexpression of inflammatory cytokines in the colonic mucosa. In conclusion, MaR1 administration ameliorates the inflammatory state in the colonic mucosa and partially compensates changes on gut microbiota caused by obesity.

Background: Fibroblast growth factor receptor (FGFR)1 and FGFR4 have been associated with tumorigenesis in a variety of tumour types. As a therapeutic approach, their inhibition has been attempted in different types of malignancies, including lung cancer, and was initially focused on FGFR1-amplified tumours, though with limited success. Methods: In vitro and in vivo functional assessments of the oncogenic potential of downregulated/overexpressed genes in isogenic cell lines were performed, as well as inhibitor efficacy tests in vitro and in vivo in patient-derived xenografts (PDXs). mRNA was extracted from FFPE non-small cell lung cancer samples to determine the prognostic potential of the genes under study. Findings: We provide in vitro and in vivo evidence showing that expression of the adhesion molecule N-cadherin is key for the oncogenic role of FGFR1/4 in non-small cell lung cancer. According to this, assessment of the expression of genes in different lung cancer patient cohorts showed that FGFR1 or FGFR4 expression alone showed no prognostic potential, and that only co-expression of FGFR1 and/or FGFR4 with N-cadherin inferred a poorer outcome. Treatment of high-FGFR1 and/or FGFR4-expressing lung cancer cell lines and patient-derived xenografts with selective FGFR inhibitors showed high efficacy, but only in models with high FGFR1/4 and N-cadherin expression. Interpretation: Our data show that the determination of the expression of FGFR1 or FGFR4 alone is not sufficient to predict anti-FGFR therapy efficacy; complementary determination of N-cadherin expression may further optimise patient selection for this therapeutic strategy. (c) 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license. (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Motivation The principal lines of research in MS/MS based Proteomics have been directed toward the molecular characterization of the proteins including their biological functions and their implications in human diseases. Recent advances in this field have also allowed the first attempts to apply these techniques to the clinical practice. Nowadays, the main progress in Computational Proteomics is based on the integration of genomic, transcriptomic and proteomic experimental data, what is known as Proteogenomics. This methodology is being especially useful for the discovery of new clinical biomarkers, small open reading frames and microproteins, although their validation is still challenging. Results We detected novel peptides following a proteogenomic workflow based on the MiTranscriptome human assembly and shotgun experiments. The annotation approach generated three custom databases with the corresponding peptides of known and novel transcripts of both protein coding genes and non-coding genes. In addition, we used a peptide detectability filter to improve the computational performance of the proteomic searches, the statistical analysis and the robustness of the results. These innovative additional filters are specially relevant when noisy next generation sequencing experiments are used to generate the databases. This resource, MiTPeptideDB, was validated using 43 cell lines for which RNA-Seq experiments and shotgun experiments were available. Availability and implementation MiTPeptideDB is available at http://bit.ly/MiTPeptideDB. Supplementary information Supplementary data are available at Bioinformatics online.

The gut microbiome has been recognized as a tool for understanding adiposity accumulation and for providing personalized nutrition advice for the management of obesity and accompanying metabolic complications. The genetic background is also involved in human energy homeostasis. In order to increase the value of nutrigenetic dietary advice, the interplay between genetics and microbiota must be investigated. The purpose of the present study was to evaluate interactive associations between gut microbiota composition and 95 obesity-related single nucleotide polymorphisms (SNPs) searched in the literature. Oral mucosa and fecal samples from 360 normal weight, overweight and obese subjects were collected. Next generation genotyping of these 95 SNPs and fecal 16S rRNA sequencing were performed. A genetic risk score (GRS) was constructed with 10 SNPs statistically or marginally associated with body mass index (BMI). Several microbiome statistical analyses at family taxonomic level were applied (LEfSe, Canonical Correspondence Analysis, MetagenomeSeq and Random Forest), and Prevotellaceae family was found in all of them as one of the most important bacterial families associated with BMI and GRS. Thus, in this family it was further analyzed the interactive association between BMI and GRS with linear regression models. Interestingly, women with higher abundance of Prevotellaceae and higher GRS were more obese, compared to women with higher GRS and lower abundance of Prevotellaceae. These findings suggest relevant interrelationships between Prevotellaceae and the genetic background that may determine interindividual BMI differences in women, which opens the way to new precision nutrition-based treatments for obesity.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found the miRNA cluster mir181ab1 as a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically-engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype underscoring their non-redundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in two highly aggressive and difficult to treat KRAS-mutated cancers.

The Human Proteome Project (HPP) is leading the international effort to characterize the human proteome. Although the main goal of this project was first focused on the detection of missing proteins, a new challenge arose from the need to assign biological functions to the uncharacterized human proteins and describe their implications in human diseases. Not only the proteins with experimental evidence (uPE1 proteins) but also the uncharacterized missing proteins (uMPs) were the objects of study in this challenge, neXt-CP50. In this work, we developed a new bioinformatic approach to infer biological annotations for the uPE1 proteins and uMPs based on a "guilt-by-association" analysis using public RNA-Seq data sets. We used the correlation of these proteins with the well-characterized PE1 proteins to construct a network. In this way, we applied the PageRank algorithm to this network to identify the most relevant nodes, which were the biological annotations of the uncharacterized proteins. All of the generated information was stored in a database. In addition, we implemented the web application UPEFinder (https://upefinder. proteored.org ) to facilitate the access to this new resource. This information is especially relevant for the researchers of the HPP who are interested in the generation and validation of new hypotheses about the functions of these proteins. Both the database and the web application are publicly available (https://github.com/tibioinformat/UPEfinder).

Type 2 diabetes (T2D) is a complex metabolic disease, which involves a maintained hyperglycemia due to the development of an insulin resistance process. Among multiple risk factors, host intestinal microbiota has received increasing attention in T2D etiology and progression. In the present study, we have explored the effect of long-term supplementation with a non-dairy fermented food product (FFP) in Zucker Diabetic and Fatty (ZDF) rats T2D model. The supplementation with FFP induced an improvement in glucose homeostasis according to the results obtained from fasting blood glucose levels, glucose tolerance test, and pancreatic function. Importantly, a significantly reduced intestinal glucose absorption was found in the FFP-treated rats. Supplemented animals also showed a greater survival suggesting a better health status as a result of the FFP intake. Some dissimilarities have been observed in the gut microbiota population between control and FFP-treated rats, and interestingly a tendency for better cardiometabolic markers values was appreciated in this group. However, no significant differences were observed in body weight, body composition, or food intake between groups. These findings suggest that FFP induced gut microbiota modifications in ZDF rats that improved glucose metabolism and protected from T2D development.

In this study we interrogated the DNA methylome of myelofibrosis patients using high-density DNA methylation arrays. We detected 35,215 differentially methylated CpG, corresponding to 10,253 genes, between myelofibrosis patients and healthy controls. These changes were present both in primary and secondary myelofibrosis, which showed no differences between them. Remarkably, most differentially methylated CpG were located outside gene promoter regions and showed significant association with enhancer regions. This aberrant enhancer hypermethylation was negatively correlated with the expression of 27 genes in the myelofibrosis cohort. Of these, we focused on the ZFP36L1 gene and validated its decreased expression and enhancer DNA hypermethylation in an independent cohort of patients and myeloid cell-lines. In vitro reporter assay and 5'-azacitidine treatment confirmed the functional relevance of hypermethylation of ZFP36L1 enhancer. Furthermore, in vitro rescue of ZFP36L1 expression had an impact on cell proliferation and induced apoptosis in SET-2 cell line indicating a possible role of ZFP36L1 as a tumor suppressor gene in myelofibrosis. Collectively, we describe the DNA methylation profile of myelofibrosis, identifying extensive changes in enhancer elements and revealing ZFP36L1 as a novel candidate tumor suppressor gene.

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (similar to 40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4.

Sunitinib is one of the most widely used targeted therapeutics for renal cell carcinoma (RCC), but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in RCC, we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and after development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in silico prediction models, six predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1, and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function renders tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the six proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

Sunitinib is one of the most widely used targeted therapeutics for renal cell-cancer (RCC) but acquired resistance against targeted therapies remains a major clinical challenge. To dissect mechanisms of acquired resistance and unravel reliable predictive biomarkers for sunitinib in renal cell-cancer (RCC), we sequenced the exons of 409 tumor-suppressor genes and oncogenes in paired tumor samples from an RCC patient, obtained at baseline and following development of acquired resistance to sunitinib. From newly arising mutations, we selected, using in-silico prediction models, 6 predicted to be deleterious, located in G6PD, LRP1B, SETD2, TET2, SYNE1 and DCC. Consistently, immunoblotting analysis of lysates derived from sunitinib-desensitized RCC cells and their parental counterparts showed marked differences in the levels and expression pattern of the proteins encoded by these genes. Our further analysis demonstrates essential roles for these proteins in mediating sunitinib cytotoxicity and shows that their loss of function render tumor cells resistant to sunitinib in vitro and in vivo. Finally, sunitinib resistance induced by continuous exposure or by inhibition of the 6 proteins was overcome by treatment with cabozantinib or a low-dose combination of lenvatinib and everolimus. Collectively, our results unravel novel markers of acquired resistance to sunitinib and clinically relevant approaches for overcoming this resistance in RCC.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G(2)-M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in down-regulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. Significance: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

Refractory or relapsed diffuse large B-cell lymphoma (DLBCL) often associates with the activated B-cell-like (ABC) subtype and genetic alterations that drive constitutive NF-kappa B activation and impair B-cell terminal differentiation. Here, we show that DNA damage response by p53 is a central mechanism suppressing the pathogenic cooperation of IKK2ca-enforced canonical NF-kappa B and impaired differentiation resulting from Blimp1 loss in ABC-DLBCL lymphomagenesis. We provide evidences that the interplay between these genetic alterations and the tumor microenvironment select for additional molecular addictions that promote lymphoma progression, including aberrant coexpression of FOXP1 and the B-cell mutagenic enzyme activation-induced deaminase, and immune evasion through major histocompatibility complex class II downregulation, PD-L1 upregulation, and T-cell exhaustion. Consistently, PD-1 blockade cooperated with anti-CD20-mediated B-cell cytotoxicity, promoting extended T-cell reactivation and antitumor specificity that improved long-term overall survival in mice. Our data support a pathogenic cooperation among NF-kappa B-driven prosurvival, genetic instability, and immune evasion mechanisms in DLBCL and provide preclinical proof of concept for including PD-1/PD-L1 blockade in combinatorial immunotherapy for ABC-DLBCL.

Ochratoxin A (OTA) is a potent rodent nephrocarcinogen; being males more sensitive than females. The objective was to study the response between sexes at gene expression level (whole genome transcriptomics) in kidneys of F344 rats treated with 0.21 or 0.50 mg/kg bw OTA for 21 days. DNA methylation analysis of selected genes was also studied (MALDI-TOF mass spectrometry). OTA-induced response was dose-dependent in males and females, although clearer in males. Females showed a higher number of altered genes than males but functional analysis revealed a higher number of significantly enriched toxicity lists in 0.21 mg/kg treated males. OTA modulated damage, signaling and metabolism related lists, as well as inflammation, proliferation and oxidative stress in both sexes. Eleven toxicity lists (damage, fibrosis, cell signaling and metabolism) were exclusively altered in males while renal safety biomarker and biogenesis of mitochondria lists were exclusively enriched in females. A high number of lists (39) were significantly enriched in both sexes. However, they contained many sex-biased OTA-modulated genes, mainly phase I and II, transporters and nuclear receptors, but also others related to cell proliferation/apoptosis. No biologically relevant changes were observed in the methylation of selected genes.

High mortality rates caused by NSCLC show the need for the identification of novel therapeutic targets. In this study we have investigated the biological effects and molecular mechanisms elicited by TMPRSS4 in NSCLC. Overexpression of TMPRSS4 in LKR13¿cells increased malignancy, subcutaneous tumor growth and multiorganic metastasis. In conditional knock-down (KD) experiments, abrogation of TMPRSS4 in H358 and H2170¿cells altered proliferation, clonogenicity, tumor engraftment and tumor growth. Reduction in S and G2/M phases of the cell cycle, decreased BrdU incorporation and increased apoptosis was also found. Transcriptomic analysis in KD cells revealed downregulation of genes involved in DNA replication, such as MCM6, TYMS and CDKN1A (p21). In patients, expression of a signature of MCM6/TYMS/TMPRSS4 genes was highly associated with poor prognosis. Downregulation of TMPRSS4 significantly increased sensitivity to chemotherapy agents. In experiments using cisplatin, apoptosis and expression of the DNA-damage marker ¿-H2A was higher in cells lacking TMPRSS4. Moreover, in vivo assays demonstrated that tumors with no TMPRSS4 were significantly more sensitive to cisplatin than controls. These results show that TMPRSS4 can be considered as a novel target in NSCLC, whose inhibition increases chemosensitivity.

With the advent of OMICs technologies, both individual research groups and consortia have spear-headed the characterization of human samples of multiple pathophysiologic origins, resulting in thousands of archived genomes and transcriptomes. Although a variety of web tools are now available to extract information from OMICs data, their utility has been limited by the capacity of nonbioinformatician researchers to exploit the information. To address this problem, we have developed CANCERTOOL, a web-based interface that aims to overcome the major limitations of public transcriptomics dataset analysis for highly prevalent types of cancer (breast, prostate, lung, and colorectal). CANCERTOOL provides rapid and comprehensive visualization of gene expression data for the gene(s) of interest in well-annotated cancer datasets. This visualization is accompanied by generation of reports customized to the interest of the researcher (e.g., editable figures, detailed statistical analyses, and access to raw data for reanalysis). It also carries out gene-to-gene correlations in multiple datasets at the same time or using preset patient groups. Finally, this new tool solves the time-consuming task of performing functional enrichment analysis with gene sets of interest using up to 11 different databases at the same time. Collectively, CANCERTOOL represents a simple and freely accessible interface to interrogate well-annotated datasets and obtain publishable representations that can contribute to refinement and guidance of cancer-related investigations at all levels of hypotheses and design.Significance: In order to facilitate access of research groups without bioinformatics support to public transcriptomics data, we have developed a free online tool with an easy-to-use interface that allows researchers to obtain quality information in a readily publishable format.

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers.

This study aims to assess methylation modifications in blood cell genes induced by eicosapentaenoic acid (EPA) and/or alpha-lipoic acid (LA) supplementation, and their potential relationship with metabolic risk biomarkers. Healthy overweight/obese women were assigned to 4 experimental groups (control or groups supplemented with 1.3 g EPA/day, 0.3 g LA/day, or both), all followed a 10-week hypocaloric diet. White blood cells DNA was hybridized in Human-450K-methylation microarray. Differentially methylated CpGs (post-pre) were identified in supplemented groups, including CpG regions from NCK2, FITM2, TRRAP, RPTOR and CREBBP genes. In peripheral blood mononuclear cells (PBMC), LA upregulated NCK2, TRRAP and RPTOR mRNA, which negatively associated with changes in body weight and fat mass. Changes in cg10320884 (TRRAP) methylation site negatively correlated with changes in TRRAP mRNA in PBMC, and positively with Framingham score. Further studies are needed to better characterize the potential involvement of epigenetics in the actions of LA and EPA. (C) 2017 Elsevier Ltd. All rights reserved.

Background: Activated protein C/endothelial protein C receptor (APC/EPCR) axis is physiologically involved in anticoagulant and cytoprotective activities in endothelial cells. Emerging evidence indicates that EPCR also plays a role in breast stemness and human tumorigenesis. Yet, its contribution to breast cancer progression and metastasis has not been elucidated. Methods: Transcriptomic status of EPCR was examined in a cohort of 286 breast cancer patients. Cell growth kinetics was evaluated in control and EPCR and SPARC/osteonectin, Cwcv, and kazal-like domains proteoglycan (SPOCK1/testican 1) silenced breast cancer cells in 2D, 3D, and in co-culture conditions. Orthotopic tumor growth and lung and osseous metastases were evaluated in several human and murine xenograft breast cancer models. Tumor-stroma interactions were further studied in vivo by immunohistochemistry and flow cytometry. An EPCR-induced gene signature was identified by microarray analysis. Results: Analysis of a cohort of breast cancer patients revealed an association of high EPCR levels with adverse clinical outcome. Interestingly, EPCR knockdown did not affect cell growth kinetics in 2D but significantly reduced cell growth in 3D cultures. Using several human and murine xenograft breast cancer models, we showed that EPCR silencing reduced primary tumor growth and secondary outgrowths at metastatic sites, including the skeleton and the lungs. Interestingly, these effects were independent of APC ligand stimulation in vitro and in vivo. Transcriptomic analysis of EPCR-silenced tumors unveiled an effect mediated by matricellular secreted proteoglycan SPOCK1/testican 1. Interestingly, SPOCK1 silencing suppressed in vitro 3D growth. Moreover, SPOCK1 ablation severely decreased orthotopic tumor growth and reduced bone metastatic osteolytic tumors. High SPOCK1 levels were also associated with poor clinical outcome in a subset breast cancer patients. Our results suggest that EPCR through SPOCK1 confers a cell growth advantage in 3D promoting breast tumorigenesis and metastasis. Conclusions: EPCR represents a clinically relevant factor associated with poor outcome and a novel vulnerability to develop combination therapies for breast cancer patients.

Vaccination induces immunostimulatory signals that are often accompanied by regulatory mechanisms such as IL-10, which control T-cell activation and inhibit vaccine-dependent antitumor therapeutic effect. Here we characterized IL10-producing cells in different tumor models treated with therapeutic vaccines. Although several cell subsets produced IL-10 irrespective of treatment, an early vaccine-dependent induction of IL-10 was detected in dendritic cells (DC). IL-10 production defined a DC population characterized by a poorly mature phenotype, lower expression of T-cell stimulating molecules and upregulation of PD-L1. These IL-10(+) DC showed impaired in vitro T-cell stimulatory capacity, which was rescued by incubation with IL-10R and PD-L1-inhibiting antibodies. In vivo IL-10 blockade during vaccination decreased the proportion of IL-10(+) DC and improved their maturation, without modifying PD-L1 expression. Similarly, PD-L1 blockade did not affect IL-10 expression. Interestingly, vaccination combined with simultaneous blockade of IL-10 and PD-L1 induced stronger immune responses, resulting in a higher therapeutic efficacy in tumor-bearing mice. These results show that vaccine-induced immunoregulatory IL-10(+) DC impair priming of antitumor immunity, suggesting that therapeutic vaccination protocols may benefit from combined targeting of inhibitory molecules expressed by this DC subset.

Antithrombotic medications target coagulation factors. Their use is associated with an increased bleeding risk. Safer drugs are needed. The heat shock protein 70 (Hsp70) exhibits antithrombotic properties that do not influence bleeding. By using murine models, we aimed to test the hypothesis that overexpressing Hsp70 with CM-695, a first in class dual inhibitor of HDAC6 and phosphodiesterase 9, protects against thrombosis while leaves bleeding tendency unaltered. CM-695 was used to induce Hsp70 overexpression. Hsp70 overexpressing mice were submitted to three thrombosis-triggering procedures. The ferric chloride carotid artery model was used to compare the antithrombotic role of CM-695 and rivaroxaban, a direct oral anticoagulant. The mouse tail transection model was used to compare the bleeding tendency upon CM-695 or rivaroxaban administration. Intraperitoneal (i.p.) 20 mg/kg CM-695 increased Hsp70 expression markedly in the murine aortic tissue. This treatment delayed thrombosis in the collagen/epinephrine [p=0.04 (Log-Rank test), n=10], Rose Bengal/laser [median vessel occlusion time (OT): 58.6 vs 39.0 minutes (min) in the control group (CG), p=0.008, n >= 10] and ferric chloride (OT: 14.7 vs 9.2 min in the CG, p=0.032, n >= 10) models. I.p. 80 mg/kg CM-695 (n >= 9) and intravenous 3 mg/kg rivaroxaban (n >= 8) significantly delayed thrombosis. CM-695 did not induce bleeding [median bleeding time (BT): 8.5 vs 7.5 min in the CG, n >= 10]. However, BT was dramatically increased by rivaroxaban (30.0 vs 13.7 min in the CG, p=0.001, n=10). In conclusion, CM-695 is a new antithrombotic small molecule devoid of bleeding risk that may be envisioned as a useful clinical tool.

The SET (I2PP2A) oncoprotein is a potent inhibitor of protein phosphatase 2A (PP2A) that regulates many cell processes and important signaling pathways. Despite the importance of SET overexpression and its prognostic impact in both hematologic and solid tumors, little is known about the mechanisms involved in its transcriptional regulation. In this report, we define the minimal promoter region of the SET gene, and identify a novel multi-protein transcription complex, composed of MYC, SP1, RUNX1 and GATA2, which activates SET expression in AML. The role of MYC is crucial, since it increases the expression of the other three transcription factors of the complex, and supports their recruitment to the promoter of SET. These data shed light on a new regulatory mechanism in cancer, in addition to the already known PP2A-MYC and SET-PP2A. Besides, we show that there is a significant positive correlation between the expression of SET and MYC, RUNX1, and GATA2 in AML patients, which further endorses our results. Altogether, this study opens new directions for understanding the mechanisms that lead to SET overexpression, and demonstrates that MYC, SP1, RUNX1 and GATA2 are key transcriptional regulators of SET expression in AML.

The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanism

Viral clearance during acute hepatitis C virus (HCV) infection is associated with the induction of potent antiviral T-cell responses. Since dendritic cells (DC) are essential in the activation of primary T-cell responses, gene expression was analyzed in DC from patients during acute HCV infection. By using microarrays, gene expression was compared in resting and activated peripheral blood plasmacytoid (pDC) and myeloid (mDC) DC from acute HCV resolving patients (AR) and from patients who become chronically infected (ANR), as well as in healthy individuals (CTRL) and chronically-infected patients (CHR). For pDC, a high number of upregulated genes was found in AR patients, irrespective of DC stimulation. However, for mDC, most evident differences were detected after DC stimulation, again corresponding to upregulated genes in AR patients. Divergent behavior of ANR was also observed when analyzing DC from CTRL and CHR, with ANR patients clustering again apart from these groups. These differences corresponded to metabolism-associated genes and genes belonging to pathways relevant for DC activation and cytokine responses. Thus, upregulation of relevant genes in DC during acute HCV infection may determine viral clearance, suggesting that dysfunctional DC may be responsible for the lack of efficient T-cell responses which lead to chronic HCV infection.

Epigenetics has an important role in the regulation of metabolic adaptation to environmental modifications. In this sense, the determination of epigenetic changes in non-invasive samples during the development of metabolic diseases could play an important role in the procedures in primary healthcare practice. To help translate the knowledge of epigenetics to public health practice, the present study aims to explore the parallelism of methylation levels between white blood cells and buccal samples in relation to obesity and associated disorders. Blood and buccal swap samples were collected from a subsample of the Spanish cohort of the Food4Me study. Infinium HumanMethylation450 DNA Analysis was carried out for the determination of methylation levels. Standard deviation for ß values method and concordance correlation analysis were used to select those CpG which showed best parallelism between samples. A total of 277 CpGs met the criteria and were selected for an enrichment analysis and a correlation analysis with anthropometrical and clinical parameters. From those selected CpGs, four presented high associations with BMI (cg01055691 in GAP43; r = -0.92 and rho = -0.84 for blood; r = -0.89 and rho = -0.83 for buccal sample), HOMA-IR (cg00095677 in ATP2A3; r = 0.82 and rho = -0.84 for blood; r = -0.8 and rho = -0.83 for buccal sample) and leptin (cg14464133 in ADARB2; r = -0.9182 and rho = -0.94 for blood; r = -0.893 and rho = -0.79 for buccal sample). These findings demonstrate the potential application of non-invasive buccal samples in the identification of surrogate epigenetic biomarkers and identify methylation sites in GAP43, ATP2A3 and ADARB2 genes as potential targets in relation to overweight management and insulin sensibility.

AIMS: Atrial fibrillation (AF) is a major risk factor for cardio-embolic stroke. Anticoagulant drugs are effective in preventing AF-related stroke. However, the high frequency of anticoagulant-associated major bleeding is a major concern. This study sought to identify new targets to develop safer antithrombotic therapies. METHODS AND RESULTS: Here, microarray analysis in peripheral blood cells in eight patients with AF and stroke and eight AF subjects without stroke brought to light a stroke-related gene expression pattern. HSPA1B, which encodes for heat-shock protein 70 kDa (Hsp70), was the most differentially expressed gene. This gene was down-regulated in stroke subjects, a finding confirmed further in an independent AF cohort of 200 individuals. Hsp70 knock-out mice subjected to different thrombotic challenges developed thrombosis significantly earlier than their wild-type (WT) counterparts. Remarkably, the tail bleeding time was unchanged. Accordingly, both TRC051384 and tubastatin A, i.e. two Hsp70 inducers via different pathways, delayed thrombus formation in WT mice, the tail bleeding time still being unaltered. Most interestingly, Hsp70 inducers did not increase the bleeding risk even when aspirin was concomitantly administered. Hsp70 induction was associated with an increased vascular thrombomodulin expression and higher circulating levels of activated protein C upon thrombotic stimulus. CONCLUSIONS: Hsp70 induction is a novel approach to delay thrombus formation with minimal bleeding risk, and is especially promising for treating AF patients and in other situations where there is also a major bleeding hazard.

For the first time an association between ACSF2 expression and the risk of recurrent DVT is suggested. Should this association be confirmed in larger prospective studies, ACSF2 could become useful for the selection of patients requiring extended anticoagulant therapy.

Stem cell-derived cardiomyocytes (CMs) are often electrophysiologically immature and heterogeneous, which represents a major barrier to their in vitro and in vivo application. Therefore, the purpose of this study was to examine whether Neuregulin-1 beta (NRG-1 beta) treatment could enhance in vitro generation of mature "working-type" CMs from induced pluripotent stem (iPS) cells and assess the regenerative effects of these CMs on cardiac tissue after acute myocardial infarction (AMI). With that purpose, adult mouse fibroblast-derived iPS from alpha-MHC-GFP mice were derived and differentiated into CMs through NRG-1 beta and/or dimethyl sulfoxide (DMSO) treatment. Cardiac specification and maturation of the iPS was analyzed by gene expression array, quantitative real-time polymerase chain reaction, immunofluorescence, electron microscopy, and patch-clamp techniques. In vivo, the iPS-derived CMs or culture medium control were injected into the peri-infarct region of hearts after coronary artery ligation, and functional and histology changes were assessed from 1 to 8 weeks post-transplantation. On differentiation, the iPS displayed early and robust in vitro cardiogenesis, expressing cardiac-specific genes and proteins. More importantly, electrophysiological studies demonstrated that a more mature ventricular-like cardiac phenotype was achieved when cells were treated with NRG-1 beta and DMSO compared with DMSO alone. Furthermore, in vivo studies demonstrated that iPS-derived CMs were able to engraft and electromechanically couple to heart tissue, ultimately preserving cardiac function and inducing adequate heart tissue remodeling. In conclusion, we have demonstrated that combined treatment with NRG-1 beta and DMSO leads to efficient differentiation of iPS into ventricular-like cardiac cells with a higher degree of maturation, which are capable of preserving cardiac function and tissue viability when transplanted into a mouse model of AMI.

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

ackground: Glioblastoma multiforme (GBM) is the most frequent malignant brain tumor in adults, and its prognosis remains dismal despite intensive research and therapeutic advances. Diagnostic biomarkers would be clinically meaningful to allow for early detection of the tumor and for those cases in which surgery is contraindicated or biopsy results are inconclusive. Recent findings show that GBM cells release microvesicles that contain a select subset of cellular proteins and RNA. The aim of this hypothesis-generating study was to assess the diagnostic potential of miRNAs found in microvesicles isolated from the serum of GBM patients. Methods: To control disease heterogeneity, we used patients with newly diagnosed GBM. In the discovery stage, PCR-based TaqMan Low Density Arrays followed by individual quantitative reverse transcriptase polymerase chain reaction were used to test the differences in the miRNA expression levels of serum microvesicles among 25 GBM patients and healthy controls paired by age and sex. The detected noncoding RNAs were then validated in another 50 GBM patients. Results: We found that the expression levels of 1 small noncoding RNA (RNU6-1) and 2 microRNAs (miR-320 and miR-574-3p) were significantly associated with a GBM diagnosis. In addition, RNU6-1 was consistently an independent predictor of a GBM diagnosis. Conclusions: Altogether our results uncovered a small noncoding RNA signature in microvesicles isolated from GBM patient serum that could be used as a fast and reliable differential diagnostic biomarker.

Purpose: To identify tissue microRNAs predictive of sunitinib activity in patients with metastatic renal-cell-carcinoma (MRCC) and to evaluate in vitro their mechanism of action in sunitinib resistance. Methods: We screened 673 microRNAs using TaqMan Low-density-Arrays (TLDAs) in tumors from MRCC patients with extreme phenotypes of marked efficacy and resistance to sunitinib, selected from an identification cohort (n = 41). The most relevant differentially expressed microRNAs were selected using bioinformatics-based target prediction analysis and quantified by qRT-PCR in tumors from patients presenting similar phenotypes selected from an independent cohort (n = 101). In vitro experiments were conducted to study the role of miR-942 in sunitinib resistance. Results: TLDAs identified 64 microRNAs differentially expressed in the identification cohort. Seven candidates were quantified by qRT-PCR in the independent series. MiR-942 was the most accurate predictor of sunitinib efficacy (p = 0.0074). High expression of miR-942, miR-628-5p, miR-133a, and miR-484 was significantly associated with decreased time to progression and overall survival. These microRNAs were also overexpressed in the sunitinib resistant cell line Caki-2 in comparison with the sensitive cell line. MiR-942 overexpression in Caki-2 up-regulates MMP-9 and VEGF secretion which, in turn, promote HBMEC endothelial migration and sunitinib resistance. Conclusions: We identified differentially expressed microRNAs in MRCC patients presenting marked sensitivity or resistance to sunitinib. MiR-942 was the best predictor of efficacy. We describe a novel paracrine mechanism through which high miR-942 levels in MRCC cells up-regulates MMP-9 and VEGF secretion to enhance endothelial migration and sunitinib resistance. Our results support further validation of these miRNA in clinical confirmatory studies.

Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor-intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome-like vesicles (ELV). Yet the functional relevance of the transference of tumor-derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown. Comparative transcriptomic profiling using an in vivo murine model of bone metastasis identified a repressed miRNA signature associated with high prometastatic activity. Forced expression of single miRNAs identified miR-192 that markedly appeased osseous metastasis in vivo, as shown by X-ray, bioluminescence imaging and microCT scans. Histological examination of metastatic lesions revealed impaired tumor-induced angiogenesis in vivo, an effect that was associated in vitro with decreased hallmarks of angiogenesis. Isolation and characterization of ELV by flow cytometry, Western blot analysis, transmission electron microscopy and nanoparticle tracking analysis revealed the ELV cargo enrichment in miR-192. Consistent with these findings, fluorescent labeled miR-192-enriched-ELV showed the in vitro transfer and release of miR-192 in target

Introduction: Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence from different in vivo liver disease models suggesting a profibrogenic role for SPARC. Methods: Two in vivo models of liver fibrosis, based on TAA administration and bile duct ligation, were developed on SPARC wild-type (SPARC(+/+)) and knock-out (SPARC(-/-)) mice. Hepatic SPARC expression was analyzed by qPCR. Fibrosis was assessed by Sirius Red staining, and the maturation state of collagen fibers was analyzed using polarized light. Necroinflammatory activity was evaluated by applying the Knodell score and liver inflammatory infiltration was characterized by immunohistochemistry. Hepatic stellate cell activation was assessed by alpha-SMA immunohistochemistry. In addition, pro-fibrogenic genes and inflammatory cytokines were measured by qPCR and/or ELISA. Liver gene expression profile was analyzed in SPARC(-/-) and SPARC(+/+) mice using Affymetrix Mouse Gene ST 1.0 array. Results: SPARC expression was found induced in fibrotic livers of mouse and human. SPARC(-/-) mice showed a reduction in the degree of inflammation, mainly CD4+ cells, and fibrosis. Consistently, collagen deposits and mRNA expression levels were decreased in SPARC(-/-) mice when compared to SPARC+/+ mice; in addition, MMP-2 expression was increased in SPARC(-/-) mice. A reduction in the number of activated myofibroblasts was observed. Moreover, TGF-beta 1 expression levels were down-regulated in the liver as well as in the serum of TAA-treated knock-out animals. Ingenuity Pathway Analysis (IPA) analysis suggested several gene networks which might involve protective mechanisms of SPARC deficiency against liver fibrogenesis and a better established machinery to repair DNA and detoxify from external chemical stimuli. Conclusions: Overall our data suggest that SPARC plays a significant role in liver fibrogenesis. Interventions to inhibit SPARC expression are suggested as promising approaches for liver fibrosis treatment.

MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction from iPSCs upon treatment with high concentrations of Activin-A. The reprogrammed iPSCs assumed an embryonic stem cell (ESC)-like miRNA signature, marked by the induction of pluripotency clusters miR-290-295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs resulted in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, analysis of the expression of these 13 upregulated miRNAs in hepatocytes and pancreatic islets revealed a tendency for these miRNAs to be expressed more in pancreatic islets than in hepatocytes. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed.

The association between inflammation and lung tumor development has been clearly demonstrated. However, little is known concerning the molecular events preceding the development of lung cancer. In this study, we characterize a chemically induced lung cancer mouse model in which lung cancer developed in the presence of silicotic chronic inflammation. Silica-induced lung inflammation increased the incidence and multiplicity of lung cancer in mice treated with N-nitrosodimethylamine, a carcinogen found in tobacco smoke. Histologic and molecular analysis revealed that concomitant chronic inflammation contributed to lung tumorigenesis through induction of preneoplastic changes in lung epithelial cells. In addition, silica-mediated inflammation generated an immunosuppressive microenvironment in which we observed increased expression of programmed cell death protein 1 (PD-1), transforming growth factor-beta 1, monocyte chemotactic protein 1 (MCP-1), lymphocyte-activation gene 3 (LAG3), and forkhead box P3 (FOXP3), as well as the presence of regulatory T cells. Finally, the K-RAS mutational profile of the tumors changed from Q61R to G12D mutations in the inflammatory milieu. In summary, we describe some of the early molecular changes associated to lung carcinogenesis in a chronic inflammatory microenvironment and provide novel information concerning the mechanisms underlying the formation and the fate of preneoplastic lesions in the silicotic lung.

The Chromosome 16 Consortium forms part of the Human Proteome Project that aims to develop an entire map of the proteins encoded by the human genome following a chromosome-centric strategy (C-HPP) to make progress in the understanding of human biology in health and disease (B/D-HPP). A Spanish consortium of 16 laboratories was organized into five working groups: Protein/Antibody microarrays, protein expression and Peptide Standard, S/MRM, Protein Sequencing, Bioinformatics and Clinical healthcare, and Biobanking. The project is conceived on a multicenter configuration, assuming the standards and integration procedures already available in ProteoRed-ISCIII, which is encompassed within HUPO initiatives. The products of the 870 protein coding genes in chromosome 16 were analyzed in Jurkat T lymphocyte cells, MCF-7 epithelial cells, and the CCD18 fibroblast cell line as it is theoretically expected that most chromosome 16 protein coding genes are expressed in at least one of these. The transcriptome and proteome of these cell lines was studied using gene expression microarray and shotgun proteomics approaches, indicating an ample coverage of chromosome 16. With regard to the B/D section, the main research areas have been adopted and a biobanking initiative has been designed to optimize methods for sample collection, management, and storage under normalized conditions and to define QC standards. The general strategy of the Chr-16 HPP and the current state of the different initiatives are discussed.

Lung cancer is a leading cause of cancer death worldwide. Several alterations in RNA metabolism have been found in lung cancer cells; this suggests that RNA metabolism-related molecules are involved in the development of this pathology. In this study, we searched for RNA metabolism-related genes that exhibit different expression levels between normal and tumor lung tissues. We identified eight genes differentially expressed in lung adenocarcinoma microarray datasets. Of these, seven were up-regulated whereas one was down-regulated. Interestingly, most of these genes had not previously been associated with lung cancer. These genes play diverse roles in mRNA metabolism: three are associated with the spliceosome (ASCL3L1, SNRPB and SNRPE), whereas others participate in RNA-related processes such as translation (MARS and MRPL3), mRNA stability (PCBPC1), mRNA transport (RAE), or mRNA editing (ADAR2, also known as ADARB1). Moreover, we found a high incidence of loss of heterozygosity at chromosome 21q22.3, where the ADAR2 locus is located, in NSCLC cell lines and primary tissues, suggesting that the downregulation of ADAR2 in lung cancer is associated with specific genetic losses. Finally, in a series of adenocarcinoma patients, the expression of five of the deregulated genes (ADAR2, MARS, RAE, SNRPB and SNRPE) correlated with prognosis. Taken together, these results support the hypothesis that changes in RNA metabolism are involved in the pathogenesis of lung cancer, and identify new potential targets for the treatment of this disease.

Background: Deregulated miRNA expression plays a crucial role in carcinogenesis. Recent studies show different mechanisms leading to miRNA deregulation in cancer; however, alterations affecting miRNAs by DNA copy number variations (CNV) remain poorly studied. Results: Our integrative analysis including data from high resolution SNPs arrays, mRNA expression arrays, and miRNAs expression profiles in 16 myeloid cell lines highlights that CNV are alternative mechanisms to deregulate the expression of miRNAs in acute myeloid leukemia (AML), and represent a novel approach to identify novel candidate genes involved in AML. We found association between the expression levels of 19 miRNAs and CNVs affecting their loci. Functional analysis showed that NF1 is a direct target of miR-370, and that overexpression of miR-370 has similar effects that NF1 inactivation, increasing proliferation and colony formation in AML cells. Moreover, real time RT-PCR showed that NF1 downregulation is a recurrent event in AML (30.8%), and western blot analysis confirmed this result. MiR-370 overexpression and deletions affecting the NF1 locus were identified as alternative mechanisms to downregulate NF1. Conclusions: NF1 downregulation is a common event in AML, and both deletions in the NF1 locus and overexpression of miR-370 are alternative mechanisms to downregulate NF1 in this disease. Our results suggest a leukemogenic role of miR-370 through NF1 downregulation in AML cells. Since NF1 deficiency leads to RAS activation, patients with AML and overexpression of miR-370 may potentially benefit from additional treatment with either RAS or mTOR inhibitors.

High-throughput gene expression technologies based on DNA microarrays allow the examination of biological systems. However, the interpretation of the complex molecular descriptions generated by these approaches is still challenging. The development of new methodologies to identify common regulatory mechanisms involved in the control of the expression of a set of co-expressed genes might enhance our capacity to extract functional information from genomic data sets. In this chapter, we describe a method that integrates different sources of information: gene expression data, genome sequence information, described transcription factor binding sites (TFBSs), functional information, and bibliographic data. The starting point of the analysis is the extraction of promoter sequences from a whole genome and the detection of TFBSs in each gene promoter. This information allows the identification of enriched TFBSs in the proximal promoter of differentially expressed genes. The functional and bibliographic interpretation of the results improves our biological insight into the regulatory mechanisms involved in a microarray experiment.

BACKGROUND: Prostate cancer (PrCa) has a high incidence in Western countries and at present, there is no cure for hormone refractory prostate cancer. Transgenic mouse models have proven useful for understanding mechanisms of prostate carcinogenesis. The characterization of genetically modified mouse PrCa models using high-throughput genomic analyses provides important information to guide appropriate experiment applications for such model. METHODS: We have analyzed the transcriptome of the hormone refractory and highly metastatic Fetal Globin-SV40/T-antigen (Ggamma-globin-Tag) transgenic mouse model for PrCa compared to normal mouse prostate tissue. Gene expression patterns found in Ggamma-globin-Tag mouse prostate tumors were compared with publicly available human localized and metastatic prostate tumors (GEO accession # GSE3325) through hierarchical cluster analysis, Pearson's rank correlation coefficient, and Self Organizing Feature Maps (SOM) analyses. RESULTS: Ggamma-globin-Tag tumors clustered closely with human metastatic tumors and gene expression patterns had a significant correlation (P < 0.01), unlike human localized primary tumors (P > 0.6). Bioinformatic analyses identified deregulated genetic pathways and networks in Ggamma-globin-Tag tumors, which displayed similarities to alterations in human PrCa. Changes in the expression of genes involved in DNA replication and repair (Rb1, p53, Myc, PCNA, DNMT3A) and growth factor signaling pathways (TGFbeta2, ERK1/2, NRas, and Notch1) are deregulated in the Ggamma-globin-Tag tumors, suggesting their key role in the oncogenic process. Identification of an enrichment of putative binding sites for transcription factors revealed eight transcription factors that may be important in Ggamma-globin-Tag carcinogenesis, including SP1, NF-Y, CREB, Elk1, and E2F. Novel genes related to microtubule regulation were also identified in Ggamma-globin-Tag tumors as potentially important candidate targets for PrCa. Overexpression of stathmin-1, whose expression was increased in human metastatic prostate tumors, was validated in Ggamma-globin-Tag tumors by immunohistochemistry. This protein belongs to the SV40/T-antigen cancer signature identified in previous studies in prostate, breast, and lung cancer mouse models. CONCLUSIONS: Our results show that the Ggamma-globin-Tag model for hormone refractory PrCa shares important features with aggressive, metastatic human PrCa. Given the role of stathmin-1 in the destabilization of microtubles and taxane resistance, the Ggamma-globin-Tag model and other SV40/T-antigen driven transgenic models may be useful for testing potential therapies directed at stathmin-1 in human prostate tumors.