Publications by authors named "Raffaele A Calogero"

73 Publications

Early stability and late random tumor progression of a HER2-positive primary breast cancer patient-derived xenograft.

Sci Rep 2021 Jan 15;11(1):1563. Epub 2021 Jan 15.

Laboratory of Immunology and Biology of Metastasis, Department of Experimental, Diagnostic and Specialty Medicine (DIMES), University of Bologna, Viale Filopanti 22, 40126, Bologna, Italy.

We established patient-derived xenografts (PDX) from human primary breast cancers and studied whether stability or progressive events occurred during long-term in vivo passages (up to 4 years) in severely immunodeficient mice. While most PDX showed stable biomarker expression and growth phenotype, a HER2-positive PDX (PDX-BRB4) originated a subline (out of 6 studied in parallel) that progressively acquired a significantly increased tumor growth rate, resistance to cell senescence of in vitro cultures, increased stem cell marker expression and high lung metastatic ability, along with a strong decrease of BCL2 expression. RNAseq analysis of the progressed subline showed that BCL2 was connected to three main hub genes also down-regulated (CDKN2A, STAT5A and WT1). Gene expression of progressed subline suggested a partial epithelial-to-mesenchymal transition. PDX-BRB4 with its progressed subline is a preclinical model mirroring the clinical paradox of high level-BCL2 as a good prognostic factor in breast cancer. Sequential in vivo passages of PDX-BRB4 chronically treated with trastuzumab developed progressive loss of sensitivity to trastuzumab while HER2 expression and sensitivity to the pan-HER tyrosine kinase inhibitor neratinib were maintained. Long-term PDX studies, even though demanding, can originate new preclinical models, suitable to investigate the mechanisms of breast cancer progression and new therapeutic approaches.
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http://dx.doi.org/10.1038/s41598-021-81085-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810859PMC
January 2021

Regenerative Reprogramming of the Intestinal Stem Cell State via Hippo Signaling Suppresses Metastatic Colorectal Cancer.

Cell Stem Cell 2020 10 29;27(4):590-604.e9. Epub 2020 Jul 29.

Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.
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http://dx.doi.org/10.1016/j.stem.2020.07.003DOI Listing
October 2020

Molecular and functional characterization of urine-derived podocytes from patients with Alport syndrome.

J Pathol 2020 Sep;252(1):88-100

Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.

Alport syndrome (AS) is a genetic disorder involving mutations in the genes encoding collagen IV α3, α4 or α5 chains, resulting in the impairment of glomerular basement membrane. Podocytes are responsible for production and correct assembly of collagen IV isoforms; however, data on the phenotypic characteristics of human AS podocytes and their functional alterations are currently limited. The evident loss of viable podocytes into the urine of patients with active glomerular disease enables their isolation in a non-invasive way. Here we isolated, immortalized, and subcloned podocytes from the urine of three different AS patients for molecular and functional characterization. AS podocytes expressed a typical podocyte signature and showed a collagen IV profile reflecting each patient's mutation. Furthermore, RNA-sequencing analysis revealed 348 genes differentially expressed in AS podocytes compared with control podocytes. Gene Ontology analysis underlined the enrichment in genes involved in cell motility, adhesion, survival, and angiogenesis. In parallel, AS podocytes displayed reduced motility. Finally, a functional permeability assay, using a podocyte-glomerular endothelial cell co-culture system, was established and AS podocyte co-cultures showed a significantly higher permeability of albumin compared to control podocyte co-cultures, in both static and dynamic conditions under continuous perfusion. In conclusion, our data provide a molecular characterization of immortalized AS podocytes, highlighting alterations in several biological processes related to extracellular matrix remodelling. Moreover, we have established an in vitro model to reproduce the altered podocyte permeability observed in patients with AS. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..
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http://dx.doi.org/10.1002/path.5496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589231PMC
September 2020

miR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis.

Prog Neurobiol 2020 07 24;190:101803. Epub 2020 Apr 24.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.

Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS. Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.
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http://dx.doi.org/10.1016/j.pneurobio.2020.101803DOI Listing
July 2020

Is an Actionable Mutation in High Grade Serous Ovarian Carcinoma.

Cells 2020 02 14;9(2). Epub 2020 Feb 14.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo, 10060 Torino, Italy.

Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a variant in PDXs from a high grade serous EOC. Allele frequencies of in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of and addiction of carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer.
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http://dx.doi.org/10.3390/cells9020442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072782PMC
February 2020

Live-animal imaging of native haematopoietic stem and progenitor cells.

Nature 2020 02 5;578(7794):278-283. Epub 2020 Feb 5.

Stem Cell Program, Boston Children's Hospital, Boston, MA, USA.

The biology of haematopoietic stem cells (HSCs) has predominantly been studied under transplantation conditions. It has been particularly challenging to study dynamic HSC behaviour, given that the visualization of HSCs in the native niche in live animals has not, to our knowledge, been achieved. Here we describe a dual genetic strategy in mice that restricts reporter labelling to a subset of the most quiescent long-term HSCs (LT-HSCs) and that is compatible with current intravital imaging approaches in the calvarial bone marrow. We show that this subset of LT-HSCs resides close to both sinusoidal blood vessels and the endosteal surface. By contrast, multipotent progenitor cells (MPPs) show greater variation in distance from the endosteum and are more likely to be associated with transition zone vessels. LT-HSCs are not found in bone marrow niches with the deepest hypoxia and instead are found in hypoxic environments similar to those of MPPs. In vivo time-lapse imaging revealed that LT-HSCs at steady-state show limited motility. Activated LT-HSCs show heterogeneous responses, with some cells becoming highly motile and a fraction of HSCs expanding clonally within spatially restricted domains. These domains have defined characteristics, as HSC expansion is found almost exclusively in a subset of bone marrow cavities with bone-remodelling activity. By contrast, cavities with low bone-resorbing activity do not harbour expanding HSCs. These findings point to previously unknown heterogeneity within the bone marrow microenvironment, imposed by the stages of bone turnover. Our approach enables the direct visualization of HSC behaviours and dissection of heterogeneity in HSC niches.
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http://dx.doi.org/10.1038/s41586-020-1971-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021587PMC
February 2020

A regulatory microRNA network controls endothelial cell phenotypic switch during sprouting angiogenesis.

Elife 2020 Jan 24;9. Epub 2020 Jan 24.

Department of Oncology, University of Turin, Candiolo, Italy.

Angiogenesis requires the temporal coordination of the proliferation and the migration of endothelial cells. Here, we investigated the regulatory role of microRNAs (miRNAs) in harmonizing angiogenesis processes in a three-dimensional in vitro model. We described a microRNA network which contributes to the observed down- and upregulation of proliferative and migratory genes, respectively. Global analysis of miRNA-target gene interactions identified two sub-network modules, the first organized in upregulated miRNAs connected with downregulated target genes and the second with opposite features. miR-424-5p and miR-29a-3p were selected for the network validation. Gain- and loss-of-function approaches targeting these microRNAs impaired angiogenesis, suggesting that these modules are instrumental to the temporal coordination of endothelial migration and proliferation. Interestingly, miR-29a-3p and its targets belong to a selective biomarker that is able to identify colorectal cancer patients who are responding to anti-angiogenic treatments. Our results provide a view of higher-order interactions in angiogenesis that has potential to provide diagnostic and therapeutic insights.
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http://dx.doi.org/10.7554/eLife.48095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299339PMC
January 2020

A computational approach based on the colored Petri net formalism for studying multiple sclerosis.

BMC Bioinformatics 2019 Dec 10;20(Suppl 6):623. Epub 2019 Dec 10.

Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy.

Background: Multiple Sclerosis (MS) is an immune-mediated inflammatory disease of the Central Nervous System (CNS) which damages the myelin sheath enveloping nerve cells thus causing severe physical disability in patients. Relapsing Remitting Multiple Sclerosis (RRMS) is one of the most common form of MS in adults and is characterized by a series of neurologic symptoms, followed by periods of remission. Recently, many treatments were proposed and studied to contrast the RRMS progression. Among these drugs, daclizumab (commercial name Zinbryta), an antibody tailored against the Interleukin-2 receptor of T cells, exhibited promising results, but its efficacy was accompanied by an increased frequency of serious adverse events. Manifested side effects consisted of infections, encephalitis, and liver damages. Therefore daclizumab has been withdrawn from the market worldwide. Another interesting case of RRMS regards its progression in pregnant women where a smaller incidence of relapses until the delivery has been observed.

Results: In this paper we propose a new methodology for studying RRMS, which we implemented in GreatSPN, a state-of-the-art open-source suite for modelling and analyzing complex systems through the Petri Net (PN) formalism. This methodology exploits: (a) an extended Colored PN formalism to provide a compact graphical description of the system and to automatically derive a set of ODEs encoding the system dynamics and (b) the Latin Hypercube Sampling with PRCC index to calibrate ODE parameters for reproducing the real behaviours in healthy and MS subjects.To show the effectiveness of such methodology a model of RRMS has been constructed and studied. Two different scenarios of RRMS were thus considered. In the former scenario the effect of the daclizumab administration is investigated, while in the latter one RRMS was studied in pregnant women.

Conclusions: We propose a new computational methodology to study RRMS disease. Moreover, we show that model generated and calibrated according to this methodology is able to reproduce the expected behaviours.
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http://dx.doi.org/10.1186/s12859-019-3196-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904991PMC
December 2019

Look for methods, not conclusions.

Cell Death Dis 2019 12 5;10(12):931. Epub 2019 Dec 5.

Department of Pharmacy, University of Salerno, Fisciano, I-84084, Italy.

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http://dx.doi.org/10.1038/s41419-019-2179-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895120PMC
December 2019

BITS2018: the fifteenth annual meeting of the Italian Society of Bioinformatics.

BMC Bioinformatics 2019 Nov 22;20(Suppl 9):562. Epub 2019 Nov 22.

Department of Physics, University of Turin, Via Pietro Giuria 1, 10126, Torino, Italy.

This preface introduces the content of the BioMed Central Bioinformatics journal Supplement related to the 15th annual meeting of the Bioinformatics Italian Society, BITS2018. The Conference was held in Torino, Italy, from June 27th to 29th, 2018.
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http://dx.doi.org/10.1186/s12859-019-3175-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873652PMC
November 2019

rCASC: reproducible classification analysis of single-cell sequencing data.

Gigascience 2019 09;8(9)

Department of Molecular Biotechnology and Health Sciences, University of Torino, Via Nizza 52, 10125 Torino, Italy.

Background: Single-cell RNA sequencing is essential for investigating cellular heterogeneity and highlighting cell subpopulation-specific signatures. Single-cell sequencing applications have spread from conventional RNA sequencing to epigenomics, e.g., ATAC-seq. Many related algorithms and tools have been developed, but few computational workflows provide analysis flexibility while also achieving functional (i.e., information about the data and the tools used are saved as metadata) and computational reproducibility (i.e., a real image of the computational environment used to generate the data is stored) through a user-friendly environment.

Findings: rCASC is a modular workflow providing an integrated analysis environment (from count generation to cell subpopulation identification) exploiting Docker containerization to achieve both functional and computational reproducibility in data analysis. Hence, rCASC provides preprocessing tools to remove low-quality cells and/or specific bias, e.g., cell cycle. Subpopulation discovery can instead be achieved using different clustering techniques based on different distance metrics. Cluster quality is then estimated through the new metric "cell stability score" (CSS), which describes the stability of a cell in a cluster as a consequence of a perturbation induced by removing a random set of cells from the cell population. CSS provides better cluster robustness information than the silhouette metric. Moreover, rCASC's tools can identify cluster-specific gene signatures.

Conclusions: rCASC is a modular workflow with new features that could help researchers define cell subpopulations and detect subpopulation-specific markers. It uses Docker for ease of installation and to achieve a computation-reproducible analysis. A Java GUI is provided to welcome users without computational skills in R.
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http://dx.doi.org/10.1093/gigascience/giz105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732171PMC
September 2019

Extracellular Vesicles Mediate Mesenchymal Stromal Cell-Dependent Regulation of B Cell PI3K-AKT Signaling Pathway and Actin Cytoskeleton.

Front Immunol 2019 12;10:446. Epub 2019 Mar 12.

Stem Cell Research Laboratory, Section of Hematology, Department of Medicine, University of Verona, Verona, Italy.

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches.
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http://dx.doi.org/10.3389/fimmu.2019.00446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423067PMC
September 2020

MicroRNAs from saliva of anopheline mosquitoes mimic human endogenous miRNAs and may contribute to vector-host-pathogen interactions.

Sci Rep 2019 02 27;9(1):2955. Epub 2019 Feb 27.

Department of Public Health and Infectious Diseases, "Sapienza" University, Piazzale Aldo Moro 5, 00185, Rome, Italy.

During blood feeding haematophagous arthropods inject into their hosts a cocktail of salivary proteins whose main role is to counteract host haemostasis, inflammation and immunity. However, animal body fluids are known to also carry miRNAs. To get insights into saliva and salivary gland miRNA repertoires of the African malaria vector Anopheles coluzzii we used small RNA-Seq and identified 214 miRNAs, including tissue-enriched, sex-biased and putative novel anopheline miRNAs. Noteworthy, miRNAs were asymmetrically distributed between saliva and salivary glands, suggesting that selected miRNAs may be preferentially directed toward mosquito saliva. The evolutionary conservation of a subset of saliva miRNAs in Anopheles and Aedes mosquitoes, and in the tick Ixodes ricinus, supports the idea of a non-random occurrence pointing to their possible physiological role in blood feeding by arthropods. Strikingly, eleven of the most abundant An. coluzzi saliva miRNAs mimicked human miRNAs. Prediction analysis and search for experimentally validated targets indicated that miRNAs from An. coluzzii saliva may act on host mRNAs involved in immune and inflammatory responses. Overall, this study raises the intriguing hypothesis that miRNAs injected into vertebrates with vector saliva may contribute to host manipulation with possible implication for vector-host interaction and pathogen transmission.
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http://dx.doi.org/10.1038/s41598-019-39880-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6393464PMC
February 2019

DNA damage and transcriptional regulation in iPSC-derived neurons from Ataxia Telangiectasia patients.

Sci Rep 2019 01 24;9(1):651. Epub 2019 Jan 24.

Department of Research, Fondazione IRCCS Istituto Nazionale Tumori, Milano, Via Amadeo 42, 20133, Milano, Italy.

Ataxia Telangiectasia (A-T) is neurodegenerative syndrome caused by inherited mutations inactivating the ATM kinase, a master regulator of the DNA damage response (DDR). What makes neurons vulnerable to ATM loss remains unclear. In this study we assessed on human iPSC-derived neurons whether the abnormal accumulation of DNA-Topoisomerase 1 adducts (Top1ccs) found in A-T impairs transcription elongation, thus favoring neurodegeneration. Furthermore, whether neuronal activity-induced immediate early genes (IEGs), a process involving the formation of DNA breaks, is affected by ATM deficiency. We found that Top1cc trapping by CPT induces an ATM-dependent DDR as well as an ATM-independent induction of IEGs and repression especially of long genes. As revealed by nascent RNA sequencing, transcriptional elongation and recovery were found to proceed with the same rate, irrespective of gene length and ATM status. Neuronal activity induced by glutamate receptors stimulation, or membrane depolarization with KCl, triggered a DDR and expression of IEGs, the latter independent of ATM. In unperturbed A-T neurons a set of genes (FN1, DCN, RASGRF1, FZD1, EOMES, SHH, NR2E1) implicated in the development, maintenance and physiology of central nervous system was specifically downregulated, underscoring their potential involvement in the neurodegenerative process in A-T patients.
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http://dx.doi.org/10.1038/s41598-018-36912-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346060PMC
January 2019

NUAK2 is a critical YAP target in liver cancer.

Nat Commun 2018 11 16;9(1):4834. Epub 2018 Nov 16.

Stem Cell Program, Boston Children's Hospital, Boston, MA, 02115, USA.

The Hippo-YAP signaling pathway is a critical regulator of proliferation, apoptosis, and cell fate. The main downstream effector of this pathway, YAP, has been shown to be misregulated in human cancer and has emerged as an attractive target for therapeutics. A significant insufficiency in our understanding of the pathway is the identity of transcriptional targets of YAP that drive its potent growth phenotypes. Here, using liver cancer as a model, we identify NUAK2 as an essential mediator of YAP-driven hepatomegaly and tumorigenesis in vivo. By evaluating several human cancer cell lines we determine that NUAK2 is selectively required for YAP-driven growth. Mechanistically, we found that NUAK2 participates in a feedback loop to maximize YAP activity via promotion of actin polymerization and myosin activity. Additionally, pharmacological inactivation of NUAK2 suppresses YAP-dependent cancer cell proliferation and liver overgrowth. Importantly, our work here identifies a specific, potent, and actionable target for YAP-driven malignancies.
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http://dx.doi.org/10.1038/s41467-018-07394-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240092PMC
November 2018

Reproducible bioinformatics project: a community for reproducible bioinformatics analysis pipelines.

BMC Bioinformatics 2018 Oct 15;19(Suppl 10):349. Epub 2018 Oct 15.

Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.

Background: Reproducibility of a research is a key element in the modern science and it is mandatory for any industrial application. It represents the ability of replicating an experiment independently by the location and the operator. Therefore, a study can be considered reproducible only if all used data are available and the exploited computational analysis workflow is clearly described. However, today for reproducing a complex bioinformatics analysis, the raw data and the list of tools used in the workflow could be not enough to guarantee the reproducibility of the results obtained. Indeed, different releases of the same tools and/or of the system libraries (exploited by such tools) might lead to sneaky reproducibility issues.

Results: To address this challenge, we established the Reproducible Bioinformatics Project (RBP), which is a non-profit and open-source project, whose aim is to provide a schema and an infrastructure, based on docker images and R package, to provide reproducible results in Bioinformatics. One or more Docker images are then defined for a workflow (typically one for each task), while the workflow implementation is handled via R-functions embedded in a package available at github repository. Thus, a bioinformatician participating to the project has firstly to integrate her/his workflow modules into Docker image(s) exploiting an Ubuntu docker image developed ad hoc by RPB to make easier this task. Secondly, the workflow implementation must be realized in R according to an R-skeleton function made available by RPB to guarantee homogeneity and reusability among different RPB functions. Moreover she/he has to provide the R vignette explaining the package functionality together with an example dataset which can be used to improve the user confidence in the workflow utilization.

Conclusions: Reproducible Bioinformatics Project provides a general schema and an infrastructure to distribute robust and reproducible workflows. Thus, it guarantees to final users the ability to repeat consistently any analysis independently by the used UNIX-like architecture.
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http://dx.doi.org/10.1186/s12859-018-2296-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191970PMC
October 2018

Development, Function, and Clinical Significance of Plasmacytoid Dendritic Cells in Chronic Myeloid Leukemia.

Cancer Res 2018 11 30;78(21):6223-6234. Epub 2018 Aug 30.

Department of Hematology, Oncology and Immunology, University Hospital Giessen and Marburg, Campus Marburg, Philipps University Marburg, Marburg, Germany.

Plasmacytoid dendritic cells (pDC) are the main producers of a key T-cell-stimulatory cytokine, IFNα, and critical regulators of antiviral immunity. Chronic myeloid leukemia (CML) is caused by BCR-ABL, which is an oncogenic tyrosine kinase that can be effectively inhibited with ABL-selective tyrosine kinase inhibitors (TKI). BCR-ABL-induced suppression of the transcription factor interferon regulatory factor 8 was previously proposed to block pDC development and compromise immune surveillance in CML. Here, we demonstrate that pDCs in newly diagnosed CML (CML-pDC) develop quantitatively normal and are frequently positive for the costimulatory antigen CD86. They originate from low-level -expressing precursors. CML-pDCs also retain their competence to maturate and to secrete IFN. RNA sequencing reveals a strong inflammatory gene expression signature in CML-pDCs. Patients with high CML-pDC counts at diagnosis achieve inferior rates of deep molecular remission (MR) under nilotinib, unless nilotinib therapy is combined with IFN, which strongly suppresses circulating pDC counts. Although most pDCs are -negative in MR, a substantial proportion of CML-pDCs persists under TKI treatment. This could be of relevance, because CML-pDCs elicit CD8 T cells, which protect wild-type mice from CML. Together, pDCs are identified as novel functional DC population in CML, regulating antileukemic immunity and treatment outcome in CML. CML-pDC originates from low-level BCR-ABL expressing stem cells into a functional immunogenic DC-population regulating antileukemic immunity and treatment outcome in CML. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-1477DOI Listing
November 2018

MicroRNA miR-34a downregulates FOXP1 during DNA damage response to limit BCR signalling in chronic lymphocytic leukaemia B cells.

Leukemia 2019 02 15;33(2):403-414. Epub 2018 Aug 15.

Molecular Medicine, CEITEC MU, Brno, Czech Republic.

The variable clinical course in chronic lymphocytic leukaemia (CLL) largely depends on p53 functionality and B-cell receptor (BCR) signalling propensity; however, it is unclear if there is any crosstalk between these pathways. We show that DNA damage response (DDR) activation leads to down-modulating the transcriptional factor FOXP1, which functions as a positive BCR signalling regulator and its high levels are associated with worse CLL prognosis. We identified microRNA (miRNA) miR-34a as the most prominently upregulated miRNA during DDR in CLL cells in vitro and in vivo during FCR therapy (fludarabine, cyclophosphamide, rituximab). MiR-34a induced by DDR activation and p53 stabilization potently represses FOXP1 expression by binding in its 3'-UTR. The low FOXP1 levels limit BCR signalling partially via derepressing BCR-inhibitory molecule CD22. We also show that low miR-34a levels can be used as a biomarker for worse response or shorter progression free survival in CLL patients treated with FCR chemoimmunotherapy, and shorter overall survival, irrespective of TP53 status. Additionally, we have developed a method for the absolute quantification of miR-34a copies and defined precise prognostic/predictive cutoffs. Overall, herein, we reveal for the first time that B cells limit their BCR signalling during DDR by down-modulating FOXP1 via DDR-p53/miR-34a axis.
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http://dx.doi.org/10.1038/s41375-018-0230-xDOI Listing
February 2019

YAP-TEAD signaling promotes basal cell carcinoma development via a c-JUN/AP1 axis.

EMBO J 2018 09 23;37(17). Epub 2018 Jul 23.

Stem Cell Program, Boston Children's Hospital, Boston, MA, USA

The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap-null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK-JUN signaling, a well-established tumor-driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug-resistant BCC.
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http://dx.doi.org/10.15252/embj.201798642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120663PMC
September 2018

p190 RhoGAP promotes contact inhibition in epithelial cells by repressing YAP activity.

J Cell Biol 2018 09 22;217(9):3183-3201. Epub 2018 Jun 22.

GI Cell Biology Research Laboratory, Boston Children's Hospital and Harvard Medical School, Boston, MA

encoding p190A RhoGAP is a cancer-associated gene with a mutation spectrum suggestive of a tumor-suppressor function. In this study, we demonstrate that loss of heterozygosity for occurs in human tumors. We sought to identify tumor-suppressor capacities for p190A RhoGAP (p190A) and its paralog p190B in epithelial cells. We reveal an essential role for p190A and p190B to promote contact inhibition of cell proliferation (CIP), a function that relies on RhoGAP activity. Unbiased mRNA sequencing analyses establish that p190A and p190B modulate expression of genes associated with the Hippo pathway. Accordingly, we determine that p190A and p190B induce CIP by repressing YAP-TEAD-regulated gene transcription through activation of LATS kinases and inhibition of the Rho-ROCK pathway. Finally, we demonstrate that loss of a single p190 paralog is sufficient to elicit nuclear translocation of YAP and perturb CIP in epithelial cells cultured in Matrigel. Collectively, our data reveal a novel mechanism consistent with a tumor-suppressor function for .
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http://dx.doi.org/10.1083/jcb.201710058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122998PMC
September 2018

microRNA profiles in urine by next-generation sequencing can stratify bladder cancer subtypes.

Oncotarget 2018 Apr 17;9(29):20658-20669. Epub 2018 Apr 17.

Italian Institute for Genomic Medicine, Turin, Italy.

Bladder cancer (BC) is the most frequent malignancy of the urinary tract with a high incidence in men and smokers. Currently, there are no non-invasive markers useful for BC diagnosis and subtypes classification that could overcome invasive procedures such as cystoscopy. Dysregulated miRNA profiles have been associated with numerous cancers, including BC. Cell-free miRNAs are abundantly present in a variety of biofluids including urine and make them promising candidates in cancer biomarker discovery. In the present study, the identification of miRNA fingerprints associated with different BC status was performed by next-generation sequencing on urine samples from 66 BC and 48 controls. Three signatures based on dysregulated miRNAs have been identified by regression models, assessing the power to discriminate different BC subtypes. Altered miRNAs according to invasiveness and grade were validated by qPCR on 112 cases and 65 controls (among which 46 cases and 16 controls were an independent group of subjects while the rest were replica samples). The area under the curve (AUC) computed including three miRNAs (miR-30a-5p, let-7c-5p and miR-486-5p) altered in all BC subtypes showed a significantly increased accuracy in the discrimination of cases and controls (AUC model = 0.70; -value = 0.01). In conclusions, the non-invasive detection in urine of a selected number of miRNAs altered in different BC subtypes could lead to an accurate early diagnosis of cancer and stratification of patients.
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http://dx.doi.org/10.18632/oncotarget.25057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945522PMC
April 2018

Luminal breast cancer-specific circular RNAs uncovered by a novel tool for data analysis.

Oncotarget 2018 03 19;9(18):14580-14596. Epub 2018 Feb 19.

Center for Molecular Systems Biology, University of Turin, Turin, Italy.

Circular RNAs are highly stable molecules present in all eukaryotes generated by distinct transcript processing. We have exploited poly(A-) RNA-Seq data generated in our lab in MCF-7 breast cancer cells to define a compilation of exonic circRNAs more comprehensive than previously existing lists. Development of a novel computational tool, named , allowed us to more accurately characterize circRNAs and to quantitatively evaluate their expression in publicly available RNA-Seq data from breast cancer cell lines and tumor tissues. We observed and confirmed, by ChIP analysis, that exons involved in circularization events display significantly higher levels of the histone post-transcriptional modification H3K36me3 than non-circularizing exons. This result has potential impact on circRNA biogenesis since H3K36me3 has been involved in alternative splicing mechanisms. By analyzing an Ago-HITS-CLIP dataset we also found that circularizing exons overlapped with an unexpectedly higher number of Ago binding sites than non-circularizing exons. Finally, we observed that a subset of MCF-7 circRNAs are specific to tumor versus normal tissue, while others can distinguish Luminal from other tumor subtypes, thus suggesting that circRNAs can be exploited as novel biomarkers and drug targets for breast cancer.
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http://dx.doi.org/10.18632/oncotarget.24522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865691PMC
March 2018

Small non-coding RNA profiling in human biofluids and surrogate tissues from healthy individuals: description of the diverse and most represented species.

Oncotarget 2018 Jan 14;9(3):3097-3111. Epub 2017 Dec 14.

Italian Institute for Genomic Medicine, IIGM (formerly Human Genetics Foundation, HuGeF), Turin, Italy.

The role of non-coding RNAs in different biological processes and diseases is continuously expanding. Next-generation sequencing together with the parallel improvement of bioinformatics analyses allows the accurate detection and quantification of an increasing number of RNA species. With the aim of exploring new potential biomarkers for disease classification, a clear overview of the expression levels of common/unique small RNA species among different biospecimens is necessary. However, except for miRNAs in plasma, there are no substantial indications about the pattern of expression of various small RNAs in multiple specimens among healthy humans. By analysing small RNA-sequencing data from 243 samples, we have identified and compared the most abundantly and uniformly expressed miRNAs and non-miRNA species of comparable size with the library preparation in four different specimens (plasma exosomes, stool, urine, and cervical scrapes). Eleven miRNAs were commonly detected among all different specimens while 231 miRNAs were globally unique across them. Classification analysis using these miRNAs provided an accuracy of 99.6% to recognize the sample types. piRNAs and tRNAs were the most represented non-miRNA small RNAs detected in all specimen types that were analysed, particularly in urine samples. With the present data, the most uniformly expressed small RNAs in each sample type were also identified. A signature of small RNAs for each specimen could represent a reference gene set in validation studies by RT-qPCR. Overall, the data reported hereby provide an insight of the constitution of the human miRNome and of other small non-coding RNAs in various specimens of healthy individuals.
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http://dx.doi.org/10.18632/oncotarget.23203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790449PMC
January 2018

Clonal analysis of lineage fate in native haematopoiesis.

Nature 2018 01 3;553(7687):212-216. Epub 2018 Jan 3.

Stem Cell Program, Boston Children's Hospital, Boston, Massachusetts 02115, USA.

Haematopoiesis, the process of mature blood and immune cell production, is functionally organized as a hierarchy, with self-renewing haematopoietic stem cells and multipotent progenitor cells sitting at the very top. Multiple models have been proposed as to what the earliest lineage choices are in these primitive haematopoietic compartments, the cellular intermediates, and the resulting lineage trees that emerge from them. Given that the bulk of studies addressing lineage outcomes have been performed in the context of haematopoietic transplantation, current models of lineage branching are more likely to represent roadmaps of lineage potential than native fate. Here we use transposon tagging to clonally trace the fates of progenitors and stem cells in unperturbed haematopoiesis. Our results describe a distinct clonal roadmap in which the megakaryocyte lineage arises largely independently of other haematopoietic fates. Our data, combined with single-cell RNA sequencing, identify a functional hierarchy of unilineage- and oligolineage-producing clones within the multipotent progenitor population. Finally, our results demonstrate that traditionally defined long-term haematopoietic stem cells are a significant source of megakaryocyte-restricted progenitors, suggesting that the megakaryocyte lineage is the predominant native fate of long-term haematopoietic stem cells. Our study provides evidence for a substantially revised roadmap for unperturbed haematopoiesis, and highlights unique properties of multipotent progenitors and haematopoietic stem cells in situ.
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http://dx.doi.org/10.1038/nature25168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884107PMC
January 2018

SeqBox: RNAseq/ChIPseq reproducible analysis on a consumer game computer.

Bioinformatics 2018 03;34(5):871-872

Department of Molecular Biotechnology and Health Sciences, University of Torino, 10124 Turin, Italy.

Summary: Short reads sequencing technology has been used for more than a decade now. However, the analysis of RNAseq and ChIPseq data is still computational demanding and the simple access to raw data does not guarantee results reproducibility between laboratories. To address these two aspects, we developed SeqBox, a cheap, efficient and reproducible RNAseq/ChIPseq hardware/software solution based on NUC6I7KYK mini-PC (an Intel consumer game computer with a fast processor and a high performance SSD disk), and Docker container platform. In SeqBox the analysis of RNAseq and ChIPseq data is supported by a friendly GUI. This allows access to fast and reproducible analysis also to scientists with/without scripting experience.

Availability And Implementation: Docker container images, docker4seq package and the GUI are available at http://www.bioinformatica.unito.it/reproducibile.bioinformatics.html.

Contact: beccuti@di.unito.it.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btx674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030956PMC
March 2018

LSD1 mediates MYCN control of epithelial-mesenchymal transition through silencing of metastatic suppressor NDRG1 gene.

Oncotarget 2017 Jan;8(3):3854-3869

Department of Biology, University of Naples 'Federico II', Naples, Italy.

Neuroblastoma (NB) with MYCN amplification is a highly aggressive and metastatic tumor in children. The high recurrence rate and resistance of NB cells to drugs urgently demands a better therapy for this disease. We have recently found that MYCN interacts with the lysine-specific demethylase 1 (LSD1), a histone modifier that participates in key aspects of gene transcription. In cancer cells, LSD1 contributes to the genetic reprogramming that underlies to Epithelial-Mesenchymal Transition (EMT) and tumor metastasis. Here, we show that LSD1 affects motility and invasiveness of NB cells by modulating the transcription of the metastasis suppressor NDRG1 (N-Myc Downstream-Regulated Gene 1). At mechanistic level, we found that LSD1 co-localizes with MYCN at the promoter region of the NDRG1 gene and inhibits its expression. Pharmacological inhibition of LSD1 relieves repression of NDRG1 by MYCN and affects motility and invasiveness of NB cells. These effects were reversed by overexpressing NDRG1. In NB tissues, high levels of LSD1 correlate with low levels of NDRG1 and reduced patients survival. Collectively, our findings elucidate a mechanism of how MYCN/LSD1 control motility and invasiveness of NB cells through transcription regulation of NDRG1 expression and suggest that pharmacological targeting of LSD1 represents a valuable approach for NB therapy.
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http://dx.doi.org/10.18632/oncotarget.12924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354800PMC
January 2017

Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk.

mBio 2015 Nov 17;6(6):e01765-15. Epub 2015 Nov 17.

R&D Centre, GSK Vaccines, Siena, Italy.

Unlabelled: The ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeable Haemophilus influenzae (NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epithelium in vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection in Haemophilus species. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies.

Importance: Simultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.
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http://dx.doi.org/10.1128/mBio.01765-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659474PMC
November 2015

YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers.

Mol Cell 2015 Oct 1;60(2):328-37. Epub 2015 Oct 1.

Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA; Harvard Stem Cell Institute, Boston, MA 02115, USA. Electronic address:

The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.
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http://dx.doi.org/10.1016/j.molcel.2015.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4624327PMC
October 2015