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Oncogenes BRAF(V600E) and SETDB1 in melanoma Transgenic zebrafish carrying the human oncogene BRAF(V600E) , the most common mutation in melanoma patients, provide a convenient model for melanoma. Two papers from Leonard Zon and colleagues demonstrate the potential of this system in the study of cancer genetics and in drug development. Ceol et al . screen for genes that cooperate with mutated BRAF , and identify SETDB1 as capable of accelerating melanoma formation in fish. The gene is found in a region that is frequently amplified in human melanomas, and its gene product, SETDB1, is a histone methylating enzyme that is often overexpressed in those melanomas. This work establishes SETDB1 as an important oncogene. White et al . find expression of a gene signature in melanoma-susceptible zebrafish embryos that is indicative of disrupted differentiation of neural crest progenitors. A chemical screen identifies leflunomide, an immunomodulatory drug used to treat rheumatoid arthritis, as an inhibitor of neural crest stem cells. Leflunomide has antimelanoma activity in human melanoma xenografts and might prove useful as an anticancer drug, particularly in combination with BRAF inhibitors. Using a zebrafish model of melanoma, this study has searched for genes that can cooperate with mutated BRAF, a frequent oncogenic event in human melanomas. It is found that SETDB1 can accelerate melanoma formation in fish and resides in a region frequently amplified in human melanomas. SETDB1, a histone methylating enzyme, is also frequently overexpressed in human melanomas and functions at least in part by regulating the expression of HOX genes. The most common mutation in human melanoma, BRAF(V600E) , activates the serine/threonine kinase BRAF and causes excessive activity in the mitogen-activated protein kinase pathway 1 , 2 . BRAF(V600E) mutations are also present in benign melanocytic naevi 3 , highlighting the importance of additional genetic alterations in the genesis of malignant tumours. Such changes include recurrent copy number variations that result in the amplification of oncogenes 4 , 5 . For certain amplifications, the large number of genes in the interval has precluded an understanding of the cooperating oncogenic events. Here we have used a zebrafish melanoma model to test genes in a recurrently amplified region of chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to accelerate melanoma formation significantly in zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing and gene expression analyses uncovered genes, including HOX genes, that are transcriptionally dysregulated in response to increased levels of SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.

Inhibitors of Vascular Endothelial Growth Factor target both tumor vasculature and cancer cells that have hijacked VEGF Receptors (VEGFRs) signaling for tumor growth-promoting activities. It is important to get precise insight in the specificity of cell responses to these antiangiogenic drugs to maximize their efficiency and minimize off-target systemic toxicity. Here we report that Axitinib, an inhibitor of VEGFRs currently in use as a second line treatment for advanced renal cell carcinoma, promotes senescence of human endothelial cells in vitro. A one-hour pulse of Axitinib is sufficient for triggering cell senescence. Mechanistically, this requires oxidative stress-dependent activation of the Ataxia Telangiectasia Mutated (ATM) kinase. Axitinib-mediated senescence promoting action is prevented by short-term treatment with antioxidants or ATM inhibitors, which conversely fail to prevent senescence induced by the DNA-damaging drug doxorubicin. Coherently, induction of oxidative stress-related genes distinguishes the response of endothelial cells to Axitinib from that to doxorubicin. Importantly, an Axitinib pulse causes cell senescence in glioblastoma cells. However, neither antioxidants nor ATM inhibitors can reverse this phenotype. Thus, antioxidants may selectively protect endothelial cells from Axitinib by decreasing systemic toxicity and maintaining a functional vascularization necessary for efficient delivery of chemotherapeutic drugs within the tumor mass.

Recent advances in pharmacogenomics have generated a wealth of data of different types whose analysis have helped in the identification of signatures of different cellular sensitivity/resistance responses to hundreds of chemical compounds. Among the different data types, gene expression has proven to be the more successful for the inference of drug response in cancer cell lines. Although effective, the whole transcriptome can introduce noise in the predictive models, since specific mechanisms are required for different drugs and these realistically involve only part of the proteins encoded in the genome. We analyzed the pharmacogenomics data of 961 cell lines tested with 265 anti-cancer drugs and developed different machine learning approaches for dissecting the genome systematically and predict drug responses using both drug-unspecific and drug-specific genes. These methodologies reach better response predictions for the vast majority of the screened drugs using tens to few hundreds genes specific to each drug instead of the whole genome, thus allowing a better understanding and interpretation of drug-specific response mechanisms which are not necessarily restricted to the drug known targets.

Caspase-8 is a key player in extrinsic apoptosis and its activity is often downregulated in cancer. However, human Caspase-8 expression is retained in some tumors, including glioblastoma (GBM), suggesting that it may support cancer growth in these contexts. GBM, the most aggressive of the gliomas, is characterized by extensive angiogenesis and by an inflammatory microenvironment that support its development and resistance to therapies. We have recently shown that Caspase-8 sustains neoplastic transformation in vitro in human GBM cell lines. Here, we demonstrate that Caspase-8, through activation of NF-kB, enhances the expression and secretion of VEGF, IL-6, IL-8, IL-1beta and MCP-1, leading to neovascularization and increased resistance to Temozolomide. Importantly, the bioinformatics analysis of microarray gene expression data derived from a set of high-grade human gliomas, shows that high Caspase-8 expression levels correlate with a worse prognosis. Cancer cells are different to normal cells in various ways. Most cancer cells, for example, delete or switch off the gene for a protein called Caspase-8. This is because this protein is best known for promoting cell death and stopping tumor cells from growing. However, some cancers keep the gene for Caspase-8 switched on including glioblastoma, the most aggressive type of brain cancer in adults. This begged the question whether this protein may in fact promote the development of tumors under certain circumstances. Glioblastomas are often highly resistant to chemotherapy and can communicate with nearby cells using proteins called cytokines to promote the formation of new blood vessels. The new blood vessel allows the tumor to readily spread into healthy brain tissue, which in turn makes it difficult for surgeons to remove all the cancerous cells. As a result, glioblastomas almost always return after surgery, and so there is strong need for new effective treatments for this type of cancer. Fianco et al. have now investigated whether Caspase-8 helps glioblastomas to grow and form new blood vessels. One common method to study human cancer cells is to inject them into mice and watch how they grow, because these experiments mimic how tumors develop in the human body. When mice were injected with human glioblastoma cells with experimentally reduced levels of Caspase-8, the cells grew poorly and did not form as many new blood vessels as unaltered glioblastoma cells. Further experiments showed that, when grown in the laboratory, glioblastoma cells with less Caspase-8 were more sensitive to a chemotherapeutic drug called temozolomide. These findings confirm that Caspase-8 does boost the growth and drug resistance of at least one cancer. When Fianco et al. analyzed clinical data from patients affected by glioblastoma, they also observed that those patients with high levels of Caspase-8 often had the worse outcomes. Previous studies conducted in white blood cells showed that Caspase-8 activated a protein complex called NF-kB, which in turn led to the cells releasing cytokines. Fianco et al. have now verified that Caspase-8 promotes NF-kB activity also in glioblastoma cells, and that this causes the cancer cells to release more cytokines. As such, these findings reveal a clear link between Caspase-8 and the formation of new blood vessels by glioblastomas. Future studies are now needed to understand why Caspase-8 promotes cell death in some cancers but the formation of new blood vessels in others. Indeed, Caspase-8 might become a target for new anticancer drugs if it is possible to inhibit its cancer-boosting activity without interfering with its ability to promote cell death.

Background In this work we aimed at sequencing and assembling the goat milk transcriptome corresponding at colostrum and 120 days of lactation. To reconstruct transcripts we used both the genome as reference, and a de novo assembly approach. Additionally, we aimed at identifying the differentially expressed genes (DEGs) between the two lactation stages and at analyzing the expression of genes involved in oligosaccharides metabolism. Results A total of 44,635 different transcripts, organized in 33,757 tentative genes, were obtained using the goat genome as reference. A significant sequence similarity match was found for 40,353 transcripts (90%) against the NCBI NT and for 35,701 (80%) against the NR databases. 68% and 69% of the de novo assembled transcripts, in colostrum and 120 days of lactation samples respectively, have a significant match with the merged transcriptome obtained using Cufflinks/Cuffmerge. CSN2, PAEP, CSN1S2, CSN3, LALBA, TPT1, FTH1, M-SAA3, SPP1, GLYCAM1, EEF1A1, CTSD, FASN, RPS29, CSN1S1, KRT19 and CHEK1 were found between the top fifteen highly expressed genes. 418 loci were differentially expressed between lactation stages, among which 207 and 122 were significantly up- and down-regulated in colostrum, respectively. Functional annotation and pathway enrichment analysis showed that in goat colostrum somatic cells predominate biological processes involved in glycolysis, carbohydrate metabolism, defense response, cytokine activity, regulation of cell proliferation and cell death, vasculature development, while in mature milk, biological process associated with positive regulation of lymphocyte activation and anatomical structure morphogenesis are enriched. The analysis of 144 different oligosaccharide metabolism-related genes showed that most of these (64%) were more expressed in colostrum than in mature milk, with eight expressed at very high levels ( SLCA3, GMSD, NME2, SLC2A1, B4GALT1, B3GNT2, NANS, HEXB ). Conclusions To our knowledge, this is the first study comparing goat transcriptome of two lactation stages: colostrum and 120 days. Our findings suggest putative differences of expression between stages and can be envisioned as a base for further research in the topic. Moreover because a higher expression of genes involved in immune defense response, carbohydrate metabolism and related to oligosaccharide metabolism was identified in colostrum we here corroborate the potential of goat milk as a natural source of lactose-derived oligosaccharides and for the development of functional foods.

Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the FMR1 gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the FMR1 gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis.

The up‐regulation of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), encoded by the OLR1 gene, plays a fundamental role in the pathogenesis of atherosclerosis. Moreover, OLR1 polymorphisms were associated with increased susceptibility to acute myocardial infarction (AMI) and coronary artery diseases (CAD). In these pathologies, the identification of therapeutic approaches that can inhibit or reduce LOX‐1 overexpression is crucial. Predictive analysis showed a putative hsa‐miR‐24 binding site in the 3′UTR of OLR1, ‘naturally’ mutated by the presence of the rs1050286 single nucleotide polymorphism (SNP). Luciferase assays revealed that miR‐24 targets OLR1 3′UTR‐G, but not 3′UTR‐A (P < 0.0005). The functional relevance of miR‐24 in regulating the expression of OLR1 was established by overexpressing miR‐24 in human cell lines heterozygous (A/G, HeLa) and homozygous (A/A, HepG2) for rs1050286 SNP. Accordingly, HeLa (A/G), but not HepG2 (A/A), showed a significant down‐regulation of OLR1 both at RNA and protein level. Our results indicate that rs1050286 SNP significantly affects miR‐24 binding affinity to the 3′UTR of OLR1, causing a more efficient post‐transcriptional gene repression in the presence of the G allele. On this basis, we considered that OLR1 rs1050286 SNP may contribute to modify OLR1 susceptibility to AMI and CAD, so ORL1 SNPs screening could help to stratify patients risk.

Structural characterization of RNAs is a dynamic field, offering many modelling possibilities. RNA secondary structure models are usually characterized by an encoding that depicts structural information of the molecule through string representations or graphs. In this work, we provide a generalization of the BEAR encoding (a context-aware structural encoding we previously developed) by expanding the set of alignments used for the construction of substitution matrices and then applying it to secondary structure encodings ranging from fine-grained to more coarse-grained representations. We also introduce a re-interpretation of the Shannon Information applied on RNA alignments, proposing a new scoring metric, the Relative Information Gain (RIG). The RIG score is available for any position in an alignment, showing how different levels of detail encoded in the RNA representation can contribute differently to convey structural information. The approaches presented in this study can be used alongside state-of-the-art tools to synergistically gain insights into the structural elements that RNAs and RNA families are composed of. This additional information could potentially contribute to their improvement or increase the degree of confidence in the secondary structure of families and any set of aligned RNAs.

Background Fragile X syndrome (FXS) is caused by CGG expansion over 200 repeats at the 5′ UTR of the FMR1 gene and subsequent DNA methylation of both the expanded sequence and the CpGs of the promoter region. This epigenetic change causes transcriptional silencing of the gene. We have previously demonstrated that 5-aza-2-deoxycytidine (5-azadC) treatment of FXS lymphoblastoid cell lines reactivates the FMR1 gene, concomitant with CpG sites demethylation, increased acetylation of histones H3 and H4 and methylation of lysine 4 on histone 3. Results In order to check the specificity of the 5-azadC-induced DNA demethylation, now we performed bisulphite sequencing of the entire methylation boundary upstream the FMR1 promoter region, which is preserved in control wild-type cells. We did not observe any modification of the methylation boundary after treatment. Furthermore, methylation analysis by MS-MLPA of PWS / AS and BWS/SRS loci demonstrated that 5-azadC treatment has no demethylating effect on these regions. Genome-wide methylation analysis through Infinium 450K (Illumina) showed no significant enrichment of specific GO terms in differentially methylated regions after 5-azadC treatment. We also observed that reactivation of FMR1 transcription lasts up to a month after a 7-day treatment and that maximum levels of transcription are reached at 10–15 days after last administration of 5-azadC. Conclusions Taken together, these data demonstrate that the demethylating effect of 5-azadC on genomic DNA is not random, but rather restricted to specific regions, if not exclusively to the FMR1 promoter. Moreover, we showed that 5-azadC has a long-lasting reactivating effect on the mutant FMR1 gene.

Nucleotides are involved in several cellular processes, ranging from the transmission of genetic information, to energy transfer and storage. Both sequence and structure based methods have been developed to predict the location of nucleotide-binding sites in proteins. Here we propose a novel methodology that leverages the observation that nucleotide-binding sites have a modular structure. Nucleotides are composed of identifiable fragments, i.e. the phosphate, the nucleobase and the carbohydrate moieties. These fragments are bound by specific structural motifs that recur in proteins of different fold. Moreover these motifs behave as modules and are found in different combinations across fold space. Our method predicts binding sites for each nucleotide fragment by comparing a query protein with a database of templates extracted from proteins of known structure. Whenever a similarity is found the fragment bound by the template is transferred on the query protein, thus identifying a putative binding site. Predictions falling inside the surface of the protein are discarded, and the remaining ones are scored using clustering and conservation. The method is able to rank as first a correct prediction in the 48%, 48% and 68% of the analyzed proteins for the nucleobase, carbohydrate and phosphate respectively, while considering the first five predictions the performances change to 71%, 65% and 86% respectively. Furthermore we attempted to reconstruct the full structure of the binding site, starting from the predicted positions of the fragments. We calculated that in the 59% of the analyzed proteins the method ranks as first a reconstructed binding site or a part of it. Finally we tested the reliability of our method in a real world case in which it has to predict nucleotide-binding sites in unbound proteins. We analyzed proteins whose structure has been solved with and without the nucleotide and observed only little variations in the method performance.