Modeling cancer drug response through drug-specific informative genes.

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.

Discovering sequence and structure landscapes in RNA interaction motifs.

RNA molecules are able to bind proteins, DNA and other small or long RNAs using information at primary, secondary or tertiary structure level. Recent techniques that use cross-linking and immunoprecipitation of RNAs can detect these interactions and, if followed by high-throughput sequencing, molecules can be analysed to find recurrent elements shared by interactors, such as sequence and/or structure motifs. Many tools are able to find sequence motifs from lists of target RNAs, while others focus on structure using different approaches to find specific interaction elements. In this work, we make a systematic analysis of RBP-RNA and RNA-RNA datasets to better characterize the interaction landscape with information about multi-motifs on the same RNAs. To achieve this goal, we updated our BEAM algorithm to combine both sequence and structure information to create pairs of patterns that model motifs of interaction. This algorithm was applied to several RNA binding proteins and ncRNAs interactors, confirming already known motifs and discovering new ones. This landscape analysis on interaction variability reflects the diversity of target recognition and underlines that often both primary and secondary structure are involved in molecular recognition.

Axitinib exposure triggers endothelial cells senescence through ROS accumulation and ATM activation.

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.

The rs17084733 variant in the KIT 3' UTR disrupts a miR-221/222 binding site in gastrointestinal stromal tumour: a sponge-like mechanism conferring disease susceptibility.

Several miRNAs are dysregulated in gastrointestinal stromal tumours (GIST), and miR-221/222 appear to have a prominent role in GIST biology. Therefore, we investigated the role of DNA variants located in miR-221/222 precursor sequences and their target KIT 3'UTR. Ninety-five polymorphisms were analysed in 115 GIST cases and 88 healthy controls. KIT 3'UTR rs17084733 and pri-miR-222 rs75246947 were found significantly associated with GIST susceptibility. Specifically, KIT rs17084733 A allele was more common in GIST, particularly in KIT wild-type (WT) patients (Padj = 0.017). rs17084733 variant is located within one of the three miR-221/222 binding sites in the KIT 3'UTR, resulting in a mismatch in this seed region. Conversely, KIT mRNA levels were lower in patients carrying the variant allele, except for KIT mutant GIST. Luciferase assay data in GIST cells, generated using a construct containing all the three miR-221/222 binding sites, are consistent with KIT mRNA levels in GIST patients. Reporter assay data, generated using a construct containing only the site encompassing rs17084733, confirmed that this is a functional variant disrupting the miR-221/222 binding site. In conclusion, this is the first study investigating the role of SNPs on miR-221/222 precursor sequences and their binding region on KIT 3'UTR in GIST. We identified the KIT variant rs17084733 as a possible novel genetic biomarker for risk of developing KIT-WT GIST. Moreover, our findings suggest the role of one of the three miR-221/222 binding sites on KIT 3'UTR as endogenous sponge, soaking up and subtracting miR-221/222 to the other two sites characterized by a higher affinity.

Caspase-8 contributes to angiogenesis and chemotherapy resistance in glioblastoma.

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.

A novel method for the identification of conserved structural patterns in RNA: From small scale to high-throughput applications.

Functional RNA regions are often related to recurrent secondary structure patterns (or motifs), which can exert their role in several different ways, particularly in dictating the interaction with RNA-binding proteins, and acting in the regulation of a large number of cellular processes. Among the available motif-finding tools, the majority focuses on sequence patterns, sometimes including secondary structure as additional constraints to improve their performance. Nonetheless, secondary structures motifs may be concurrent to their sequence counterparts or even encode a stronger functional signal. Current methods for searching structural motifs generally require long pipelines and/or high computational efforts or previously aligned sequences. Here, we present BEAM (BEAr Motif finder), a novel method for structural motif discovery from a set of unaligned RNAs, taking advantage of a recently developed encoding for RNA secondary structure named BEAR (Brand nEw Alphabet for RNAs) and of evolutionary substitution rates of secondary structure elements. Tested in a varied set of scenarios, from small- to large-scale, BEAM is successful in retrieving structural motifs even in highly noisy data sets, such as those that can arise in CLIP-Seq or other high-throughput experiments.

c-MYC inhibition impairs hypoxia response in glioblastoma multiforme.

The c-MYC oncoprotein is a DNA binding transcription factor that enhances the expression of many active genes. c-MYC transcriptional signatures vary according to the transcriptional program defined in each cell type during differentiation. Little is known on the involvement of c-MYC in regulation of gene expression programs that are induced by extracellular cues such as a changing microenvironment. Here we demonstrate that inhibition of c-MYC in glioblastoma multiforme cells blunts hypoxia-dependent glycolytic reprogramming and mitochondria fragmentation in hypoxia. This happens because c-MYC inhibition alters the cell transcriptional response to hypoxia and finely tunes the expression of a subset of Hypoxia Inducible Factor 1-regulated genes. We also show that genes whose expression in hypoxia is affected by c-MYC inhibition are able to distinguish the Proneural subtype of glioblastoma multiforme, thus potentially providing a molecular signature for this class of tumors that are the least tractable among glioblastomas.

The human rs1050286 polymorphism alters LOX-1 expression through modifying miR-24 binding.

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.

Role of CTCF protein in regulating FMR1 locus transcription.

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.

Functional characterization and expression analysis of novel alternative splicing isoforms of Olr1 gene during mouse embryogenesis.

LOX-1 (Lectin-like oxidized low-density lipoprotein receptor-1) is the primary endothelial receptor of oxidized LDL (oxLDL). Both in vitro and in vivo experiments have shown this protein to be important in the initiation of atherosclerosis and to be up-regulated by pro-atherogenic factors. Recently, it has been demonstrated that Olr1, the gene encoding Lox-1, is important for tumor growth and for maintaining the transformed state in different cancer cell lines, suggesting that it acts in a molecular pathway connecting cancer and atherosclerosis. Both diseases in humans are characterized by uncontrolled regulation of cellular growth and differentiation. We present evidence that Olr1 is expressed during mouse embryogenesis in developmental stages (from 7.5 to 9.5 dpc) in which cardiogenesis occurs. In addition, we identify two novel Olr1 isoform (hereafter referred to as D3D5Olr1 and D2D5Olr1) whose spatio-temporal expression pattern overlaps with Olr1 in vivo. In vitro, D3D5Olr1 localizes to the cell surface membrane as Olr1, in contrast with D2D5Olr1; these data suggest that D2D5Olr1 isoform translates a receptor that does not reach the plasma membrane. Accordingly, in silico transmembrane protein topology prediction analyses, show that D2D5Olr1 does not contain any transmembrane region. Finally, both isoforms can activate the same genetic pathways underlying Olr1 expression, such as, hypoxia and inflammation, even if with a different efficiency. All these data suggest a new functional involvement of Olr1, and probably of its spliceforms, in murine cardiogenesis and angiogenesis.

The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.

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. BRAF(V600E) mutations are also present in benign melanocytic naevi, 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. 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.