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The view of long noncoding RNAs as nonfunctional "garbage" has been definitely outdated by the large body of evidence indicating this class of ncRNAs as "golden junk", especially in precision oncology. Indeed, in light of their oncogenic role and the higher expression in multiple cancer types compared with paired adjacent tissues, the clinical interest for lncRNAs as diagnostic and/or prognostic biomarkers has been rapidly increasing. The emergence of large-scale sequencing technologies, their subsequent diffusion even in small research and clinical centers, the technological advances for the detection of low-copy lncRNAs in body fluids, coupled to the huge reduction of operating costs, have nowadays made possible to rapidly and comprehensively profile them in multiple tumors and large cohorts. In this review, we first summarize some relevant data about the oncogenic role of well-studied lncRNAs having a clinical relevance. Then, we focus on the description of their potential use as diagnostic/prognostic biomarkers, including an updated overview about licensed patents or clinical trials on lncRNAs in oncology.

Background B-raf inhibitors (BRAFi) are effective for BRAF-mutated papillary (PTC) and anaplastic (ATC) thyroid carcinomas, although acquired resistance impairs tumour cells’ sensitivity and/or limits drug efficacy. Targeting metabolic vulnerabilities is emerging as powerful approach in cancer. Methods In silico analyses identified metabolic gene signatures and Hif-1α as glycolysis regulator in PTC. BRAF-mutated PTC, ATC and control thyroid cell lines were exposed to HIF1A siRNAs or chemical/drug treatments (CoCl2, EGF, HGF, BRAFi, MEKi and diclofenac). Genes/proteins expression, glucose uptake, lactate quantification and viability assays were used to investigate the metabolic vulnerability of BRAF-mutated cells. Results A specific metabolic gene signature was identified as a hallmark of BRAF-mutated tumours, which display a glycolytic phenotype, characterised by enhanced glucose uptake, lactate efflux and increased expression of Hif-1α-modulated glycolytic genes. Indeed, Hif-1α stabilisation counteracts the inhibitory effects of BRAFi on these genes and on cell viability. Interestingly, targeting metabolic routes with BRAFi and diclofenac combination we could restrain the glycolytic phenotype and synergistically reduce tumour cells’ viability. Conclusion The identification of a metabolic vulnerability of BRAF-mutated carcinomas and the capacity BRAFi and diclofenac combination to target metabolism open new therapeutic perspectives in maximising drug efficacy and reducing the onset of secondary resistance and drug-related toxicity.

RET rearrangements as well as BRAF and RAS mutations drive differential pathway activation in papillary thyroid carcinomas, leading to different tumor phenotypes and prognoses. Although The Cancer Genome Atlas Consortium has identified tumor subgroups based on protein-coding gene signatures, neither expression of long noncoding RNAs (lncRNA) nor their correlation with specific tumor-driving mutations and rearrangements have been systematically assessed. Here, we reanalyzed our RNA-sequencing data using a de novo discovery approach to identify lncRNAs and define tumor subtype-specific signatures of annotated lncRNAs. Among them, we identified COMET (Correlated-to-MET), a natural antisense transcript that was highly expressed in carcinomas harboring BRAFV600E mutation or RET gene rearrangements (i.e., BRAF-like tumors) and induced the downstream MAPK pathway. In papillary thyroid carcinomas, COMET was part of a coexpression network including different oncogenes belonging to the MAPK pathway, and its expression highly correlated with MET expression. Depletion of COMET resulted in reduced expression of genes within this network, including the MET oncogene. COMET repression inhibited viability and proliferation of tumor cells harboring BRAFV600E somatic mutation or RET oncogene rearrangement and dramatically reduced motility and invasiveness of tumor cells. Moreover, silencing COMET markedly increased sensitivity to vemurafenib, a common inhibitor of mutated B-raf. Collectively, our results suggest COMET as a new target to improve drug-based cancer therapies, especially in BRAF-mutated and MET-addicted papillary thyroid carcinomas.Significance:These results highlight the oncogenic role of lncRNA COMET in thyroid and indicate it as a potential new target to overcome vemurafenib resistance in BRAF-mutated and MET-addicted carcinomas.

Subtype-specific signature of protein coding genes in papillary thyroid carcinoma.

Table S1: List of oligonucleotide pairs. Table S2: COMET-correlated genes.

Legends of the Supplementary Figures

Glioblastoma multiforme (GBM) is a fatal brain tumor without effective drug treatment. In this study, we highlight, for the first time, the contribution of chromatin remodeling gene Lysine (K)-specific demethylase 5C (

Simple Summary Current treatments for complex diseases, including cancer, are generally characterized by high toxicity due to their low selectivity for target cells. Moreover, patients often develop drug resistance, hence becoming less sensitive to the therapy. For this reason, novel, improved, and more specific pharmacological therapies are needed. The high cost and the time required to develop new drugs poses the attention on the development of computational methods for drug repositioning and combination therapy prediction. In this study, we developed an integrated network pharmacology framework that combines mechanistic and chemocentric approaches in order to predict potential drug combinations for cancer therapy. We applied our paradigm in five cancer types, which we used as case studies. Our strategy can be applied to the study of any complex disease by guiding the prioritization of drug combinations. Despite remarkable efforts of computational and predictive pharmacology to improve therapeutic strategies for complex diseases, only in a few cases have the predictions been eventually employed in the clinics. One of the reasons behind this drawback is that current predictive approaches are based only on the integration of molecular perturbation of a certain disease with drug sensitivity signatures, neglecting intrinsic properties of the drugs. Here we integrate mechanistic and chemocentric approaches to drug repositioning by developing an innovative network pharmacology strategy. We developed a multilayer network-based computational framework integrating perturbational signatures of the disease as well as intrinsic characteristics of the drugs, such as their mechanism of action and chemical structure. We present five case studies carried out on public data from The Cancer Genome Atlas, including invasive breast cancer, colon adenocarcinoma, lung squamous cell carcinoma, hepatocellular carcinoma and prostate adenocarcinoma. Our results highlight paclitaxel as a suitable drug for combination therapy for many of the considered cancer types. In addition, several non-cancer-related genes representing unusual drug targets were identified as potential candidates for pharmacological treatment of cancer.

Pancreatic stellate cells (PSCs) secrete high levels of transforming growth factor-β1 (TGF-β1) that contributes to the development of pancreatic ductal adenocarcinoma (PDAC). TGF-β1 modulates the expression of L1 cell adhesion molecule (L1CAM), but its role in tumour progression still remains controversial. To clarify L1 function in PDAC and cellular phenotypes, we performed L1CAM cell sorting, silencing and overexpression in several primary pancreatic cancer cells. PSCs silenced for TGF-β1 were used for crosstalk experiments. We found that TGF-β1 secreted by PSCs negatively regulates L1CAM expression, through canonical TGF-β-Smad2/3 signalling, leading to a more aggressive PDAC phenotype. Cells with reduced expression of L1CAM harboured enhanced stemness potential and tumourigenicity. Inactivation of TGF-β1 signalling in PSCs strongly reduced the aggressiveness of PDAC cells. Our data provide functional proof and mechanistic insights for the tumour-suppressive function of L1CAM via reducing stemness. Rescuing L1CAM expression in cancer cells through targeting of TGF-β1 reverses stemness and bears the potential to improve the still miserable prognosis of PDAC patients.

X-linked lissencephaly with abnormal genitalia (XLAG) and developmental epileptic encephalopathy-1 (DEE1) are caused by mutations in the Aristaless-related homeobox (ARX) gene, which encodes a transcription factor responsible for brain development. It has been unknown whether the phenotypically diverse XLAG and DEE1 phenotypes may converge on shared pathways. To address this question, a label-free quantitative proteomic approach was applied to the neonatal brain of Arx knockout (ArxKO/Y) and knock-in polyalanine (Arx(GCG)7/Y) mice that are respectively models for XLAG and DEE1. Gene ontology and protein–protein interaction analysis revealed that cytoskeleton, protein synthesis and splicing control are deregulated in an allelic-dependent manner. Decreased α-tubulin content was observed both in Arx mice and Arx/alr-1(KO) Caenorhabditis elegans ,and a disorganized neurite network in murine primary neurons was consistent with an allelic-dependent secondary tubulinopathy. As distinct features of Arx(GCG)7/Y mice, we detected eIF4A2 overexpression and translational suppression in cortex and primary neurons. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in synaptic remodelling, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose that the allelic-dependent secondary synaptopathy results from an aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by null and polyalanine expanded mutations in ARX. The identification of these effects can aid in the design of pathway-guided therapy for ARX endophenotypes and NDDs with overlapping comorbidities.

Diagnosing a complex genetic syndrome and correctly assigning the concomitant phenotypic traits to a well-defined clinical form is often a medical challenge. In this work, we report the analysis of a family with complex phenotypes, including microcephaly, intellectual disability, dysmorphic features, and polydactyly in the proband, with the aim of adding new aspects for obtaining a clear diagnosis. We performed array-comparative genomic hybridization and quantitative reverse transcriptase PCR (qRT-PCR) analyses. We identified a deletion of chromosome 20p12.1 involving the macrodomain containing 2/mono-ADP ribosylhydrolase 2 gene (MACROD2) in several members of the family. This gene is actually not associated with a specific syndrome but with congenital anomalies of multiple organs. qRT-PCR showed higher levels of a MACROD2 mRNA isoform in the individuals carrying the deletion. Our results, together with other data reported in the literature, support the hypothesis that the deletion in MACROD2 can affect correct embryonic development and that the presence of another associated event, such as epigenetic modifications at the MACROD2 locus, can influence the level of severity of the pathology.

Purpose of Review Full and partial synthetic agonists targeting the transcription factor PPAR? are contained in FDA-approved insulin-sensitizing drugs and used for the treatment of metabolic syndrome-related dysfunctions. Here, we discuss the association between PPARG genetic variants and drug efficacy, as well as the role of alternative splicing and post-translational modifications as contributors to the complexity of PPAR? signaling and to the effects of synthetic PPAR? ligands. Recent Findings PPAR? regulates the transcription of several target genes governing adipocyte differentiation and glucose and lipid metabolism, as well as insulin sensitivity and inflammatory pathways. These pleiotropic functions confer great relevance to PPAR? in physiological regulation of whole-body metabolism, as well as in the etiology of metabolic disorders. Accordingly, PPARG gene mutations, nucleotide variations, and post-translational modifications have been associated with adipose tissue disorders and the related risk of insulin resistance and type 2 diabetes (T2D). Moreover, PPAR? alternative splicing isoforms-- generating dominant-negative isoforms mainly expressed in human adipose tissue--have been related to impaired PPAR? activity and adipose tissue dysfunctions. Thus, multiple regulatory levels that contribute to PPAR? signaling complexity may account for the beneficial as well as adverse effects of PPAR? agonists. Further targeted analyses, taking into account all these aspects, are needed for better deciphering the role of PPAR? in human pathophysiology, especially in insulin resistance and T2D. Summary The therapeutic potential of full and partial PPAR? synthetic agonists underlines the clinical significance of this nuclear receptor. PPARG mutations, polymorphisms, alternative splicing isoforms, and post-translational modifications may contribute to the pathogenesis of metabolic disorders, also influencing the responsiveness of pharmacological therapy. Therefore, in the context of the current evidence-based trend to personalized diabetes management, we highlight the need to decipher the intricate regulation of PPAR? signaling to pave the way to tailored therapies in patients with insulin resistance and T2D.

Background: Next-generation sequencing (NGS) needs to be validated and standardized to ensure that cancer patients are reliably selected for target treatments. In Italy, NGS is performed in several institutions and harmonization of wet and dry procedures is needed. To this end, a consortium of five different laboratories, covering the most part of the Italian peninsula, was constituted. A narrow gene panel (SiRe®) covering 568 clinically relevant mutations in six different genes (EGFR, KRAS, NRAS, BRAF, cKIT, and PDGFRα) with a predictive role for therapy selection in non-small cell lung cancer (NSCLC), gastrointestinal stromal tumor, colorectal carcinoma (CRC), and melanoma was evaluated in each participating laboratory. Methods: To assess the NGS inter-laboratory concordance, the SiRe® panel, with a related kit and protocol for library preparation, was used in each center to analyze a common set of 20 NSCLC and CRC routine samples. Concordance rate, in terms of mutation detected and relative allelic frequencies, was assessed. Then, each institution prospectively analyzed an additional set of 40 routine samples (for a total of 160 specimens) to assess the reproducibility of the NGS run parameters in each institution. Results: An inter-laboratory agreement of 100% was reached in analyzing the data obtained from the 20 common sample sets; the concordance rate of allelic frequencies distribution was 0.989. The prospective analysis of the run metric parameters obtained by each center locally showed that the analytical performance of the SiRe® panel in the different institutions was highly reproducible. Conclusions: The SiRe® panel represents a robust diagnostic tool to harmonize the NGS procedure in different Italian laboratories.

Long noncoding RNAs (lncRNA) are emerging as players in physiological processes and as crucial contributors to pathologic states, especially cancer. The direct contribution of lncRNAs to tumorigenesis has been demonstrated by loss- or gain-of-function experiments. In this chapter, we describe the most convincing evidences about their contribution to cancer hallmarks, with a focus on lncRNAs able to promote or suppress tumor formation either by their intrinsic properties or by their capacity to modulate known oncogenes/suppressors. Herein, we also describe the main lncRNAs-targeting approaches, discussing the main pros and cons of these methodologies. We further describe preclinical models widely used to address the potential role of lncRNAs as both prognostic/diagnostic markers and therapeutic targets in the field of clinical oncology.

Reduced neo-adipogenesis and dysfunctional lipid-overloaded adipocytes are hallmarks of hypertrophic obesity linked to insulin resistance. Identifying molecular features of hypertrophic adipocytes requires appropriate in vitro models. We describe the generation of a model of human hypertrophic-like adipocytes directly comparable to normal adipose cells and the pathologic evolution toward hypertrophic state. We generate in vitro hypertrophic cells from mature adipocytes, differentiated from human mesenchymal stem cells. Combining optical, confocal, and transmission electron microscopy with mRNA/protein quantification, we characterize this cellular model, confirming specific alterations also in subcutaneous adipose tissue. Specifically, we report the generation and morphological/molecular characterization of human normal and hypertrophic-like adipocytes. The latter displays altered morphology and unbalance between canonical and dominant negative (PPARG&Delta, 5) transcripts of PPARG, paralleled by reduced expression of PPAR&gamma, targets, including GLUT4. Furthermore, the unbalance of PPAR&gamma, isoforms associates with GLUT4 down-regulation in subcutaneous adipose tissue of individuals with overweight/obesity or impaired glucose tolerance/type 2 diabetes, but not with normal weight or glucose tolerance. In conclusion, the hypertrophic-like cells described herein are an innovative tool for studying molecular dysfunctions in hypertrophic obesity and the unbalance between PPAR&gamma, isoforms associates with down-regulation of GLUT4 and other PPAR&gamma, targets, representing a new hallmark of hypertrophic adipocytes.

According to the US National Cancer Institute (NCI; http://www.cancer.gov/ accession October, 2017), bladder cancer (BC) is the 6th most common type of cancer in the Western countries, with ~350,000 new cases/year and a constantly increasing incidence (1). Patients with BC have been commonly stratified according to molecular alterations (i.e., somatic mutations and gene fusions) in FGFR3 and TP53 genes. However, large-scale studies from international consortia (especially from The Cancer Genome Atlas Research Network, Nature 2014) have depicted a more complex picture, reporting the deregulation of a large network of genes (2). Non-coding RNAs, both long and small-RNAs (lncRNAs and miRNAs, respectively)--have been postulated to contribute, at least partially, to the wide deregulation observed in BC.

RET rearrangements as well as BRAF and RAS mutations drive differential pathway activation in papillary thyroid carcinomas, leading to different tumor phenotypes and prognoses. Although The Cancer Genome Atlas Consortium has identified tumor subgroups based on protein-coding gene signatures, neither expression of long noncoding RNAs (lncRNA) nor their correlation with specific tumor-driving mutations and rearrangements have been systematically assessed. Here, we reanalyzed our RNA-sequencing data using a de novo discovery approach to identify lncRNAs and define tumor subtype-specific signatures of annotated lncRNAs. Among them, we identified COMET (Correlated-to-MET), a natural antisense transcript that was highly expressed in carcinomas harboring BRAFV600E mutation or RET gene rearrangements (i.e., BRAF-like tumors) and induced the downstream MAPK pathway. In papillary thyroid carcinomas, COMET was part of a coexpression network including different oncogenes belonging to the MAPK pathway, and its expression highly correlated with MET expression. Depletion of COMET resulted in reduced expression of genes within this network, including the MET oncogene. COMET repression inhibited viability and proliferation of tumor cells harboring BRAFV600E somatic mutation or RET oncogene rearrangement and dramatically reduced motility and invasiveness of tumor cells. Moreover, silencing COMET markedly increased sensitivity to vemurafenib, a common inhibitor of mutated B-raf. Collectively, our results suggest COMET as a new target to improve drug-based cancer therapies, especially in BRAF-mutated and MET-addicted papillary thyroid carcinomas. Significance: These results highlight the oncogenic role of lncRNA COMET in thyroid and indicate it as a potential new target to overcome vemurafenib resistance in BRAF-mutated and MET-addicted carcinomas.

Diagnosing a complex genetic syndrome and correctly assigning the concomitant phenotypic traits to a well-defined clinical form is often a medical challenge. In this work, we report the analysis of a family with complex phenotypes, including microcephaly, intellectual disability, dysmorphic features, and polydactyly in the proband, with the aim of adding new aspects for obtaining a clear diagnosis. We performed array-comparative genomic hybridization and quantitative reverse transcriptase PCR (qRT-PCR) analyses. We identified a deletion of chromosome 20p12.1 involving the macrodomain containing 2/mono-ADP ribosylhydrolase 2 gene (MACROD2) in several members of the family. This gene is actually not associated with a specific syndrome but with congenital anomalies of multiple organs. qRT-PCR showed higher levels of a MACROD2 mRNA isoform in the individuals carrying the deletion. Our results, together with other data reported in the literature, support the hypothesis that the deletion in MACROD2 can affect correct embryonic development and that the presence of another associated event, such as epigenetic modifications at the MACROD2 locus, can influence the level of severity of the pathology.

Simple Summary DNA regions having high sequence similarity among human, rat and mouse genomes are defined as Ultraconserved Regions. Non-coding RNA transcripts originating by these regions may play relevant roles in the onset and progression of multiple cancer types. We recently found that ultra-conserved-transcript-8+ (uc.8+) levels correlate with the grading and staging of bladder cancer. The aim of this study is to systematically evaluate the expression of ultra-conserved-transcript-8+ (uc.8+) in biopsies and assess its intracellular localization. Furthermore, we aimed to correlate uc.8+ levels with clinical parameters and patient survival. Our analysis indicates that uc.8+ can localize both in the cytoplasm and nucleus of bladder cells at early stages of tumorigenesis, while in tumors at advanced stages, uc.8+ has a prevalent cytoplasmic localization. These data provide relevant information about uc.8+ localization as a hallmark of tumor stage. Finally, using advanced computer-based techniques, we predicted the binding of uc.8+ to RNA-binding proteins. Our study overall suggests that uc.8+ localization can be used as a prognostic biomarker for bladder cancer. Abstract Non-coding RNA transcripts originating from Ultraconserved Regions (UCRs) have tissue-specific expression and play relevant roles in the pathophysiology of multiple cancer types. Among them, we recently identified and characterized the ultra-conserved-transcript-8+ (uc.8+), whose levels correlate with grading and staging of bladder cancer. Here, to validate uc.8+ as a potential biomarker in bladder cancer, we assessed its expression and subcellular localization by using tissue microarray on 73 human bladder cancer specimens. We quantified uc.8+ by in-situ hybridization and correlated its expression levels with clinical characteristics and patient survival. The analysis of subcellular localization indicated the simultaneous presence of uc.8+ in the cytoplasm and nucleus of cells from the Low-Grade group, whereas a prevalent cytoplasmic localization was observed in samples from the High-Grade group, supporting the hypothesis of uc.8+ nuclear-to-cytoplasmic translocation in most malignant tumor forms. Moreover, analysis of uc.8+ expression and subcellular localization in tumor-surrounding stroma revealed a marked down-regulation of uc.8+ levels compared to the paired (adjacent) tumor region. Finally, deep machine-learning approaches identified nucleotide sequences associated with uc.8+ localization in nucleus and/or cytoplasm, allowing to predict possible RNA binding proteins associated with uc.8+, recognizing also sequences involved in mRNA cytoplasm-translocation. Our model suggests uc.8+ subcellular localization as a potential prognostic biomarker for bladder cancer.

Background A novel prediction algorithm is needed for the identification of effective tumor associated mutated neoantigens. Only those with no homology to self wild type antigens are true predicted neoantigens (TPNAs) and can elicit an antitumor T cell response, not attenuated by central tolerance. To this aim, the mutational landscape was evaluated in HCV-associated hepatocellular carcinoma. Methods Liver tumor biopsies and adjacent non-tumor liver tissues were obtained from 9 HCV-chronically infected subjects and subjected to RNA-Seq analysis. Mutant peptides were derived from single nucleotide variations and TPNAs were predicted using two prediction servers (e.g. NetTepi and NetMHCstabpan) by comparison with corresponding wild-type sequences, non-related self and pathogen-related antigens. Immunological confirmation was obtained in preclinical as well as clinical setting. Results The development of such an improved algorithm resulted in a handful of TPNAs despite the large number of predicted neoantigens. Furthermore, TPNAs may share homology to pathogen’s antigens and be targeted by a pre-existing T cell immunity. Cross-reactivity between such antigens was confirmed in an experimental pre-clinical setting. Finally, TPNAs homologous to pathogen’s antigens were found in the only HCC long-term survival patient, suggesting a correlation between the pre-existing T cell immunity specific for these TPNAs and the favourable clinical outcome. Conclusions The new algorithm allowed the identification of the very few TPNAs in cancer cells, and those targeted by a pre-existing immunity strongly correlated with long-term survival. Only such TPNAs represent the optimal candidates for immunotherapy strategies. Electronic supplementary material The online version of this article (10.1186/s12967-018-1662-9) contains supplementary material, which is available to authorized users.

RNA-binding proteins may play a critical role in gene regulation in various diseases or biological processes by controlling post-transcriptional events such as polyadenylation, splicing, and mRNA stabilization via binding activities to RNA molecules. Due to the importance of RNA-binding proteins in gene regulation, a great number of studies have been conducted, resulting in a large amount of RNA-Seq datasets. However, these datasets usually do not have structured organization of metadata, which limits their potentially wide use. To bridge this gap, the metadata of a comprehensive set of publicly available mouse RNA-Seq datasets with perturbed RNA-binding proteins were collected and integrated into a database called RBPMetaDB. This database contains 278 mouse RNA-Seq datasets for a comprehensive list of 163 RNA-binding proteins. These RNA-binding proteins account for only ∼10% of all known RNA-binding proteins annotated in Gene Ontology, indicating that most are still unexplored using high-throughput sequencing. This negative information provides a great pool of candidate RNA-binding proteins for biologists to conduct future experimental studies. In addition, we found that DNA-binding activities are significantly enriched among RNA-binding proteins in RBPMetaDB, suggesting that prior studies of these DNA- and RNA-binding factors focus more on DNA-binding activities instead of RNA-binding activities. This result reveals the opportunity to efficiently reuse these data for investigation of the roles of their RNA-binding activities. A web application has also been implemented to enable easy access and wide use of RBPMetaDB. It is expected that RBPMetaDB will be a great resource for improving understanding of the biological roles of RNA-binding proteins.Database URL: http://rbpmetadb.yubiolab.org

Papillary thyroid carcinoma (PTC) is the most frequent thyroid malignant neoplasia. Oncogene activation occurs in more than 70% of the cases. Indeed, about 40% of PTCs harbor mutations in BRAF gene, whereas RET rearrangements (RET/PTC oncogenes) are present in about 20% of cases. Finally, RAS mutations and TRK rearrangements account for about 5% each of these malignancies. We used RNA-Sequencing to identify fusion transcripts and mutations in cancer driver genes in a cohort of 18 PTC patients. Furthermore, we used targeted DNA sequencing to validate identified mutations. We extended the screening to 50 PTC patients and 30 healthy individuals. Using this approach we identified new missense mutations in CBL, NOTCH1, PIK3R4 and SMARCA4 genes. We found somatic mutations in DICER1, MET and VHL genes, previously found mutated in other tumors, but not described in PTC. We identified a new chimeric transcript generated by the fusion of WNK1 and B4GALNT3 genes, correlated with B4GALNT3 overexpression. Our data confirmed PTC genetic heterogeneity, revealing that gene expression correlates more with the mutation pattern than with tumor staging. Overall, this study provides new data about mutational landscape of this neoplasia, suggesting potential pharmacological adjuvant therapies against Notch signaling and chromatin remodeling enzymes.

The filamentous bacteriophage fd, codisplaying antigenic determinants and a single chain antibody fragment directed against the dendritic cell receptor DEC-205, is a promising vaccine candidate for its safety and its ability to elicit innate and adaptive immune response in absence of adjuvants. By using a system vaccinology approach based on RNA-Sequencing (RNA-Seq) analysis, we describe a relevant gene modulation in dendritic cells pulsed with anti-DEC-205 bacteriophages fd. RNA-Seq data analysis indicates that the bacteriophage fd virions are sensed as a pathogen by dendritic cells; they activate the danger receptors that trigger an innate immune response and thus confer a strong adjuvanticity that is needed to obtain a long-lasting adaptive immune response.

Vaccination is the most successful and cost-effective method to prevent infectious diseases. However, many vaccine antigens have poor in vivo immunogenic potential and need adjuvants to enhance immune response. The application of systems biology to immunity and vaccinology has yielded crucial insights about how vaccines and adjuvants work. We have previously characterized two safe and powerful delivery systems derived from non-pathogenic prokaryotic organisms: E2 and fd filamentous bacteriophage systems. They elicit an in vivo immune response inducing CD8+ T-cell responses, even in absence of adjuvants or stimuli for dendritic cells' maturation. Nonetheless, a systematic and comparative analysis of the complex gene expression network underlying such activation is missing. Therefore, we compared the transcriptomes of ex vivo isolated bone marrow-derived dendritic cells exposed to these antigen delivery systems. Significant differences emerged, especially for genes involved in innate immunity, co-stimulation, and cytokine production. Results indicate that E2 drives polarization toward the Th2 phenotype, mainly mediated by Irf4, Ccl17, and Ccr4 over-expression. Conversely, fd-scalphaDEC-205 triggers Th1 T cells' polarization through the induction of Il12b, Il12rb, Il6, and other molecules involved in its signal transduction. The data analysis was performed using RNASeqGUI, hence, addressing the increasing need of transparency and reproducibility of computational analysis.

Ultraconserved regions (UCRs) represent a relatively new class of non-coding genomic sequences highly conserved between human, rat and mouse genomes. These regions can reside within exons of protein-coding genes, despite the vast majority of them localizes within introns or intergenic regions. Several studies have undoubtedly demonstrated that most of these regions are actively transcribed in normal cells/tissues, where they contribute to regulate many cellular processes. Interestingly, these non-coding RNAs exhibit aberrant expression levels in human cancer cells and their expression profiles have been used as prognostic factors in human malignancies, as well as to unambiguously distinguish among distinct cancer types. In this review, we first describe their identification, then we provide some updated information about their genomic localization and classification. More importantly, we discuss about the available literature describing an overview of the mechanisms through which some transcribed UCRs (T-UCR) contribute to cancer progression or to the metastatic spread. To date, the interplay between T-UCRs and microRNAs is the most convincing evidence linking T-UCRs and tumorigenesis. The limitations of these studies and the future challenges to be addressed in order to understand the biological role of T-UCRs are also discussed herein. We envision that future efforts are needed to convincingly include this class of ncRNAs in the growing area of cancer therapeutics.

The nuclear receptor PPARγis a key regulator of adipogenesis, and alterations of its function are associated with different pathological processes related to metabolic syndrome. We recently identified twoPPARGtranscripts encoding dominant negative PPARγisoforms. The existence of differentPPARGvariants suggests that alternative splicing is crucial to modulate PPARγfunction, underlying some underestimated aspects of its regulation. Here we investigatePPARGexpression in different tissues and cells affected in metabolic syndrome and, in particular, during adipocyte differentiation of human mesenchymal stem cells. We defined the transcript-specific expression pattern ofPPARGvariants encoding both canonical and dominant negative isoforms and identified a novelPPARGtranscript,γ1ORF4. Our analysis indicated that, during adipogenesis, the transcription of alternativePPARGvariants is regulated in a time-specific manner through differential usage of distinct promoters. In addition, our analysis describes—for the first time—the differential contribution of three ORF4 variants to this process, suggesting a still unexplored role for these dominant negative isoforms during adipogenesis. Therefore, our results highlight crucial aspects ofPPARGregulation, suggesting the need of further investigation to rule out the differential impact of allPPARGtranscripts in both physiologic and pathologic conditions, such as metabolism-related disorders.

A novel two-step bioinformatics strategy was applied for identification of signatures with therapeutic implications in hepatitis-associated HCC. Transcriptional profiles from HBV- and HCV-associated HCC samples were compared with non-tumor liver controls. Resulting HCC modulated genes were subsequently compared with different non-tumor tissue samples. Two related signatures were identified, namely “HCC-associated” and “HCC-specific”. Expression data were validated by RNA-Seq analysis carried out on unrelated HCC samples and protein expression was confirmed according to The Human Protein Atlas" (http://proteinatlas.org/), a public repository of immunohistochemistry data. Among all, aldo-keto reductase family 1 member B10, and IGF2 mRNA-binding protein 3 were found strictly HCC-specific with no expression in 18/20 normal tissues. Target peptides for vaccine design were predicted for both proteins associated with the most prevalent HLA-class I and II alleles. The described novel strategy showed to be feasible for identification of HCC-specific proteins as highly potential target for HCC immunotherapy.

Background/Objectives:The genomic bases of the adipose tissue abnormalities induced by chronic positive calorie excess have been only partially elucidated. We adopted a genome-wide approach to directly test whether long-term high-fat diet (HFD) exposure affects the DNA methylation profile of the mouse adipose tissue and to identify the functional consequences of these changes.Subjects/Methods:We have used epididymal fat of mice fed either high-fat (HFD) or regular chow (STD) diet for 5 months and performed genome-wide DNA methylation analyses by methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mouse Homeobox (Hox) Gene DNA Methylation PCR, RT-qPCR and bisulphite sequencing analyses were then performed.Results:Mice fed the HFD progressively expanded their adipose mass accompanied by a significant decrease in glucose tolerance (P