Forcato M, Nicoletti C, Pal K, Livi CM, Ferrari F, Bicciato S.
Comparison of computational methods for Hi-C data analysis.
Nat Methods. 2017 Jun 12 Abstract
Hi-C is a genome-wide sequencing technique used to investigate 3D chromatin conformation inside the nucleus. Computational methods are required to analyze Hi-C data and identify chromatin interactions and topologically associating domains (TADs) from genome-wide contact probability maps. We quantitatively compared the performance of 13 algorithms in their analyses of Hi-C data from six landmark studies and simulations. This comparison revealed differences in the performance of methods for chromatin interaction identification, but more comparable results for TAD detection between algorithms.
Romano O, Cifola I, Poletti V, Severgnini M, Peano C, De Bellis G, Mavilio F, Miccio A.
Retroviral Scanning: Mapping MLV Integration Sites to Define Cell-specific Regulatory Regions.
J Vis Exp. 2017 May 28 Abstract
Moloney murine leukemia (MLV) virus-based retroviral vectors integrate predominantly in acetylated enhancers and promoters. For this reason, mLV integration sites can be used as functional markers of active regulatory elements. Here, we present a retroviral scanning tool, which allows the genome-wide identification of cell-specific enhancers and promoters. Briefly, the target cell population is transduced with an mLV-derived vector and genomic DNA is digested with a frequently cutting restriction enzyme. After ligation of genomic fragments with a compatible DNA linker, linker-mediated polymerase chain reaction (LM-PCR) allows the amplification of the virus-host genome junctions. Massive sequencing of the amplicons is used to define the mLV integration profile genome-wide. Finally, clusters of recurrent integrations are defined to identify cell-specific regulatory regions, responsible for the activation of cell-type specific transcriptional programs. The retroviral scanning tool allows the genome-wide identification of cell-specific promoters and enhancers in prospectively isolated target cell populations. Notably, retroviral scanning represents an instrumental technique for the retrospective identification of rare populations (e.g. somatic stem cells) that lack robust markers for prospective isolation.
Mancarella C, Casanova-Salas I, Calatrava A, García-Flores M, Garofalo C, Grilli A, Rubio-Briones J, Scotlandi K, López-Guerrero J.
Insulin-like growth factor 1 receptor affects the survival of primary prostate cancer patients depending on TMPRSS2-ERG status.
BMC Cancer. 2017 May 25;17(1):367. doi: 10.1186/s12885-017-3356-8. Abstract
Prostate cancer (PCa) is characterized by clinical and biological heterogeneity and has differential outcomes and mortality rates. Therefore, it is necessary to identify molecular alterations to define new therapeutic strategies based on the risk of progression. In this study, the prognostic relevance of the insulin-like growth factor (IGF) system was examined in molecular subtypes defined by TMPRSS2-ERG (T2E) gene fusion within a series of patients with primary localized PCa.
A cohort of 270 formalin-fixed and paraffin-embedded (FFPE) primary PCa samples from patients with more than 5 years’ follow-up was collected. IGF-1R, IGF-1, IGFBP-3 and INSR expression was analyzed using quantitative RT-PCR. The T2E status and immunohistochemical ERG findings were considered in the analyses. The association with both biochemical and clinical progression-free survival (BPFS and PFS, respectively) was evaluated for the different molecular subtypes using the Kaplan-Meier proportional risk log-rank test and the Cox proportional hazards model.
An association between IGF-1R overexpression and better BPFS was found in T2E-negative patients (35.3% BPFS, p-value = 0.016). Multivariate analysis demonstrated that IGF-1R expression constitutes an independent variable in T2E-negative patients [HR: 0.41. CI 95% (0.2-0.82), p = 0.013]. These data were confirmed using immunohistochemistry of ERG as subrogate of T2E. High IGF-1 expression correlated with prolonged BPFS and PFS independent of the T2E status.
IGF-1R, a reported target of T2E, constitutes an independent factor for good prognosis in T2E-negative PCa. Quantitative evaluation of IGF-1/IGF-1R expression combined with molecular assessment of T2E status or ERG protein expression represents a useful marker for tumor progression in localized PCa.
Puccio S, Grillo G, Licciulli F, Severgnini M, Liuni S, Bicciato S, De Bellis G, Ferrari F, Peano C.
WoPPER: Web server for Position Related data analysis of gene Expression in Prokaryotes.
Nucleic Acids Res. 2017 Apr 29 Abstract
The structural and conformational organization of chromosomes is crucial for gene expression regulation in eukaryotes and prokaryotes as well. Up to date, gene expression data generated using either microarray or RNA-sequencing are available for many bacterial genomes. However, differential gene expression is usually investigated with methods considering each gene independently, thus not taking into account the physical localization of genes along a bacterial chromosome. Here, we present WoPPER, a web tool integrating gene expression and genomic annotations to identify differentially expressed chromosomal regions in bacteria. RNA-sequencing or microarray-based gene expression data are provided as input, along with gene annotations. The user can select genomic annotations from an internal database including 2780 bacterial strains, or provide custom genomic annotations. The analysis produces as output the lists of positionally related genes showing a coordinated trend of differential expression. Graphical representations, including a circular plot of the analyzed chromosome, allow intuitive browsing of the results. The analysis procedure is based on our previously published R-package PREDA. The release of this tool is timely and relevant for the scientific community, as WoPPER will fill an existing gap in prokaryotic gene expression data analysis and visualization tools. WoPPER is open to all users and can be reached at the following URL: https://WoPPER.ba.itb.cnr.it.
Mondanelli G, Albini E, Pallotta MT, Volpi C, Chatenoud L, Kuhn C, Fallarino F, Matino D, Belladonna ML, Bianchi R, Vacca C, Bicciato S, Boon L, Ricci G, Grohmann U, Puccetti P, Orabona C.
The Proteasome Inhibitor Bortezomib Controls Indoleamine 2,3-Dioxygenase 1 Breakdown and Restores Immune Regulation in Autoimmune Diabetes.
Front Immunol. 2017 Apr 13 Abstract
Bortezomib (BTZ) is a first-in-class proteasome inhibitor approved for the therapy of multiple myeloma that also displays unique regulatory activities on immune cells. The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme exerting potent immunoregulatory effects when expressed in dendritic cells (DCs), the most potent antigen-presenting cells capable of promoting either immunity or tolerance. We previously demonstrated that, in inflammatory conditions, IDO1 is subjected to proteasomal degradation in DCs, turning these cells from immunoregulatory to immunostimulatory. In non-obese diabetic (NOD) mice, an experimental model of autoimmune diabetes, we also identified an IDO1 defect such that the DCs do not develop tolerance toward pancreatic islet autoantigens. We found that BTZ rescues IDO1 protein expression in vitro in a particular subset of DCs, i.e., plasmacytoid DCs (pDCs) from NOD mice. When administered in vivo to prediabetic mice, the drug prevented diabetes onset through IDO1- and pDC-dependent mechanisms. Although the drug showed no therapeutic activity when administered alone to overtly diabetic mice, its combination with otherwise suboptimal dosages of autoimmune-preventive anti-CD3 antibody resulted in disease reversal in 70% diabetic mice, a therapeutic effect similar to that afforded by full-dosage anti-CD3. Thus, our data indicate a potential for BTZ in the immunotherapy of autoimmune diabetes and further underline the importance of IDO1-mediated immune regulation in such disease.
Mondanelli G, Bianchi R, Pallotta MT, Orabona C, Albini E, Iacono A, Belladonna ML, Vacca C, Fallarino F, Macchiarulo A, Ugel S, Bronte V, Gevi F, Zolla L, Verhaar A, Peppelenbosch M, Mazza EM, Bicciato S, Laouar Y, Santambrogio L, Puccetti P, Volpi C, Grohmann U.
A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells.
Immunity. 2017 Feb 21;46(2):233-244. doi: 10.1016/j.immuni.2017.01.005. Epub 2017 Feb 14. Abstract
Arginase 1 (Arg1) and indoleamine 2,3-dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l-arginine and l-tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non-enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1-dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid-derived suppressor cells, conditioned DCs toward an IDO1-dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1-phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings.
Sorrentino G, Ruggeri N, Zannini A, Ingallina E, Bertolio R, Marotta C, Neri C, Cappuzzello E, Forcato M, Rosato A, Mano M, Bicciato S, Del Sal G.
Glucocorticoid receptor signalling activates YAP in breast cancer.
Nat Commun. 2017 Jan 19;8:14073. doi: 10.1038/ncomms14073. Abstract
The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance.
Lo Sardo F, Forcato M, Sacconi A, Capaci V, Zanconato F, Di Agostino S, Del Sal G, Pandolfi PP, Strano S, Bicciato S, Blandino G.
MCM7 and its hosted miR-25, 93 and 106b cluster elicit YAP/TAZ oncogenic activity in lung cancer.
Carcinogenesis. 2017 Jan;38(1):64-75. doi: 10.1093/carcin/bgw110. Epub 2016 Oct 17. Abstract
Lung cancer is the first cause of cancer death worldwide and the Hippo pathway transcriptional coactivators YAP/TAZ have a pro-oncogenic role in this context. In order to understand the mechanisms through which YAP/TAZ elicit their oncogenic role in different systems, many studies are focused on YAP/TAZ target genes involved in the regulation of cell proliferation/survival and migration. However, there is scarce evidence on the role of YAP/TAZ in microRNA regulation while there is increasing evidence supporting the role of microRNAs in the main oncogenic processes. Here, we showed that YAP/TAZ were able to regulate several microRNAs in non-small cell lung cancer (NSCLC) cell lines. In detail, we focused on a cluster of three oncogenic microRNAs (miR-25, 93 and 106b) hosted in the MCM7 gene that were overexpressed in lung tumors compared to normal tissues. In addition, similar behavior was observed in breast cancer and head and neck tumor casuistries, where they showed a prognostic role. In NSCLC cells, YAP/TAZ induced the transcription of the MCM7 gene and its hosted miRs, thereby promoting cell proliferation through the post-transcriptional inhibition of the p21 cell cycle regulator. Accordingly, p21 was maintained at low levels in lung tumors compared to normal tissues. Conversely, its expression was restored in NSCLC cells upon YAP/TAZ interference or upon treatment with the statin cerivastatin. In summary, we provide evidence for a novel mechanism of modulation supporting the protumorigenic functions of the YAP/TAZ factors through the modulation of a bioncogenic locus consisting of one gene and three hosted microRNAs.
Marigo I, Zilio S, Desantis G, Mlecnik B, Agnellini AH, Ugel S, Sasso MS, Qualls JE, Kratochvill F, Zanovello P, Molon B, Ries CH, Runza V, Hoves S, Bilocq AM, Bindea G, Mazza EM, Bicciato S, Galon J, Murray PJ, Bronte V.
T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells.
Cancer Cell. 2016 Oct 10;30(4):651. doi: 10.1016/j.ccell.2016.09.009. Abstract
Effective cancer immunotherapy requires overcoming immunosuppressive tumor microenvironments. We found that local nitric oxide (NO) production by tumor-infiltrating myeloid cells is important for adoptively transferred CD8(+) cytotoxic T cells to destroy tumors. These myeloid cells are phenotypically similar to inducible nitric oxide synthase (NOS2)- and tumor necrosis factor (TNF)-producing dendritic cells (DC), or Tip-DCs. Depletion of immunosuppressive, colony stimulating factor 1 receptor (CSF-1R)-dependent arginase 1(+) myeloid cells enhanced NO-dependent tumor killing. Tumor elimination via NOS2 required the CD40-CD40L pathway. We also uncovered a strong correlation between survival of colorectal cancer patients and NOS2, CD40, and TNF expression in their tumors. Our results identify a network of pro-tumor factors that can be targeted to boost cancer immunotherapies.
Panciera T, Azzolin L, Fujimura A, Di Biagio D, Frasson C, Bresolin S, Soligo S, Basso G, Bicciato S, Rosato A, Cordenonsi M, Piccolo S.
Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ.
Cell Stem Cell. 2016 Dec 1;19(6):725-737. doi: 10.1016/j.stem.2016.08.009. Epub 2016 Sep 15. Abstract
The ability to induce autologous tissue-specific stem cells in culture could have a variety of applications in regenerative medicine and disease modeling. Here we show that transient expression of exogenous YAP or its closely related paralogue TAZ in primary differentiated mouse cells can induce conversion to a tissue-specific stem/progenitor cell state. Differentiated mammary gland, neuronal, and pancreatic exocrine cells, identified using a combination of cell sorting and lineage tracing approaches, efficiently convert to proliferating cells with properties of stem/progenitor cells of their respective tissues after YAP induction. YAP-induced mammary stem/progenitor cells show molecular and functional properties similar to endogenous MaSCs, including organoid formation and mammary gland reconstitution after transplantation. Because YAP/TAZ function is also important for self-renewal of endogenous stem cells in culture, our findings have implications for understanding the molecular determinants of the somatic stem cell state.
Manara MC, Terracciano M, Mancarella C, Sciandra M, Guerzoni C, Pasello M, Grilli A, Zini N, Picci P, Colombo MP, Morrione A, Scotlandi K.
CD99 triggering induces methuosis of Ewing sarcoma cells through IGF-1R/RAS/Rac1 signaling.
Oncotarget. 2016 Nov 29;7(48):79925-79942. doi: 10.18632/oncotarget.13160. Abstract
CD99 is a cell surface molecule that has emerged as a novel target for Ewing sarcoma (EWS), an aggressive pediatric bone cancer. This report provides the first evidence of methuosis in EWS, a non-apoptotic form of cell death induced by an antibody directed against the CD99 molecule. Upon mAb triggering, CD99 induces an IGF-1R/RAS/Rac1 complex, which is internalized into RAB5-positive endocytic vacuoles. This complex is then dissociated, with the IGF-1R recycling to the cell membrane while CD99 and RAS/Rac1 are sorted into immature LAMP-1-positive vacuoles, whose excessive accumulation provokes methuosis. This process, which is not detected in CD99-expressing normal mesenchymal cells, is inhibited by disruption of the IGF-1R signaling, whereas enhanced by IGF-1 stimulation. Induction of IGF-1R/RAS/Rac1 was also observed in the EWS xenografts that respond to anti-CD99 mAb, further supporting the role of the IGF/RAS/Rac1 axis in the hyperstimulation of macropinocytosis and selective death of EWS cells. Thus, we describe a vulnerability of EWS cells, including those resistant to standard chemotherapy, to a treatment with anti-CD99 mAb, which requires IGF-1R/RAS signaling but bypasses the need for their direct targeting. Overall, we propose CD99 targeting as new opportunity to treat EWS patients resistant to canonical apoptosis-inducing agents.
Magistri M, Khoury N, Mazza EM, Velmeshev D, Lee JK, Bicciato S, Tsoulfas P, Faghihi MA.
A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells.
Eur J Neurosci. 2016 Nov;44(10):2858-2870. doi: 10.1111/ejn.13382. Epub 2016 Sep 25. Abstract
Astrocytes are a morphologically and functionally heterogeneous population of cells that play critical roles in neurodevelopment and in the regulation of central nervous system homeostasis. Studies of human astrocytes have been hampered by the lack of specific molecular markers and by the difficulties associated with purifying and culturing astrocytes from adult human brains. Human neural progenitor cells (NPCs) with self-renewal and multipotent properties represent an appealing model system to gain insight into the developmental genetics and function of human astrocytes, but a comprehensive molecular characterization that confirms the validity of this cellular system is still missing. Here we used an unbiased transcriptomic analysis to characterize in vitro culture of human NPCs and to define the gene expression programs activated during the differentiation of these cells into astrocytes using FBS or the combination of CNTF and BMP4. Our results demonstrate that in vitro cultures of human NPCs isolated during the gliogenic phase of neurodevelopment mainly consist of radial glial cells (RGCs) and glia-restricted progenitor cells. In these cells the combination of CNTF and BMP4 activates the JAK/STAT and SMAD signaling cascades, leading to the inhibition of oligodendrocytes lineage commitment and activation of astrocytes differentiation. On the other hand, FBS-derived astrocytes have properties of reactive astrocytes. Our work suggests that in vitro culture of human NPCs represents a valuable cellular system to study human disorders characterized by impairment of astrocytes development and function. Our datasets represent an important resource for researchers studying human astrocytes development and might set the basis for the discovery of novel human-specific astrocyte markers.
Ronchetti D, Manzoni M, Agnelli L, Vinci C, Fabris S, Cutrona G, Matis S, Colombo M, Galletti S, Taiana E, Recchia AG, Bossio S, Gentile M, Musolino C, Di Raimondo F, Grilli A, Bicciato S, Cortelezzi A, Tassone P, Morabito F, Ferrarini M, Neri A
lncRNA profiling in early-stage chronic lymphocytic leukemia identifies transcriptional fingerprints with relevance in clinical outcome.
Blood Cancer J. 2016 Sep 9;6(9):e468. doi: 10.1038/bcj.2016.77. Abstract
Long non-coding RNAs (lncRNAs) represent a novel class of functional RNA molecules with an important emerging role in cancer. To elucidate their potential pathogenetic role in chronic lymphocytic leukemia (CLL), a biologically and clinically heterogeneous neoplasia, we investigated lncRNAs expression in a prospective series of 217 early-stage Binet A CLL patients and 26 different subpopulations of normal B-cells, through a custom annotation pipeline of microarray data. Our study identified a 24-lncRNA-signature specifically deregulated in CLL compared with the normal B-cell counterpart. Importantly, this classifier was validated on an independent data set of CLL samples. Belonging to the lncRNA signature characterizing distinct molecular CLL subgroups, we identified lncRNAs recurrently associated with adverse prognostic markers, such as unmutated IGHV status, CD38 expression, 11q and 17p deletions, and NOTCH1 mutations. In addition, correlation analyses predicted a putative lncRNAs interplay with genes and miRNAs expression. Finally, we generated a 2-lncRNA independent risk model, based on lnc-IRF2-3 and lnc-KIAA1755-4 expression, able to distinguish three different prognostic groups in our series of early-stage patients. Overall, our study provides an important resource for future studies on the functions of lncRNAs in CLL, and contributes to the discovery of novel molecular markers with clinical relevance associated with the disease.
Calabria E, Mazza EM, Dyar KA, Pogliaghi S, Bruseghini P, Morandi C, Salvagno GL, Gelati M, Guidi GC, Bicciato S, Schiaffino S, Schena F, Capelli C.
Aging: a portrait from gene expression profile in blood cells.
Aging (Albany NY). 2016 Aug;8(8):1802-21. doi: 10.18632/aging.101016. Abstract
The availability of reliable biomarkers of aging is important not only to monitor the effect of interventions and predict the timing of pathologies associated with aging but also to understand the mechanisms and devise appropriate countermeasures. Blood cells provide an easily available tissue and gene expression profiles from whole blood samples appear to mirror disease states and some aspects of the aging process itself. We report here a microarray analysis of whole blood samples from two cohorts of healthy adult and elderly subjects, aged 43±3 and 68±4 years, respectively, to monitor gene expression changes in the initial phase of the senescence process. A number of significant changes were found in the elderly compared to the adult group, including decreased levels of transcripts coding for components of the mitochondrial respiratory chain, which correlate with a parallel decline in the maximum rate of oxygen consumption (VO2max), as monitored in the same subjects. In addition, blood cells show age-related changes in the expression of several markers of immunosenescence, inflammation and oxidative stress. These findings support the notion that the immune system has a major role in tissue homeostasis and repair, which appears to be impaired since early stages of the aging process.
Moretti I, Ciciliot S, Dyar KA, Abraham R, Murgia M, Agatea L, Akimoto T, Bicciato S, Forcato M, Pierre P, Uhlenhaut NH, Rigby PW, Carvajal JJ, Blaauw B, Calabria E, Schiaffino S.
MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity.
Nat Commun. 2016 Aug 3;7:12397. doi: 10.1038/ncomms12397. Abstract
The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.
Romano O, Peano C, Tagliazucchi GM, Petiti L, Poletti V, Cocchiarella F, Rizzi E, Severgnini M, Cavazza A, Rossi C, Pagliaro P, Ambrosi A, Ferrari G, Bicciato S, De Bellis G, Mavilio F, Miccio A.
Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment.
Sci Rep. 2016 Apr 20;6:24724. doi: 10.1038/srep24724. Abstract
Genome-wide approaches allow investigating the molecular circuitry wiring the genetic and epigenetic programs of human somatic stem cells. Hematopoietic stem/progenitor cells (HSPC) give rise to the different blood cell types; however, the molecular basis of human hematopoietic lineage commitment is poorly characterized. Here, we define the transcriptional and epigenetic profile of human HSPC and early myeloid and erythroid progenitors by a combination of Cap Analysis of Gene Expression (CAGE), ChIP-seq and Moloney leukemia virus (MLV) integration site mapping. Most promoters and transcripts were shared by HSPC and committed progenitors, while enhancers and super-enhancers consistently changed upon differentiation, indicating that lineage commitment is essentially regulated by enhancer elements. A significant fraction of CAGE promoters differentially expressed upon commitment were novel, harbored a chromatin enhancer signature, and may identify promoters and transcribed enhancers driving cell commitment. MLV-targeted genomic regions co-mapped with cell-specific active enhancers and super-enhancers. Expression analyses, together with an enhancer functional assay, indicate that MLV integration can be used to identify bona fide developmentally regulated enhancers. Overall, this study provides an overview of transcriptional and epigenetic changes associated to HSPC lineage commitment, and a novel signature for regulatory elements involved in cell identity.
Cavazza A, Miccio A, Romano O, Petiti L, Malagoli Tagliazucchi G, Peano C, Severgnini M, Rizzi E, De Bellis G, Bicciato S, Mavilio F.
Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation.
Stem Cell Reports. 2016 Apr 12;6(4):618-32. doi: 10.1016/j.stemcr.2016.03.003. Epub 2016 Mar 31. Abstract
Human skin is maintained by the differentiation and maturation of interfollicular stem and progenitors cells. We used DeepCAGE, genome-wide profiling of histone modifications and retroviral integration analysis, to map transcripts, promoters, enhancers, and super-enhancers (SEs) in prospectively isolated keratinocytes and transit-amplifying progenitors, and retrospectively defined keratinocyte stem cells. We show that >95% of the active promoters are in common and differentially regulated in progenitors and differentiated keratinocytes, while approximately half of the enhancers and SEs are stage specific and account for most of the epigenetic changes occurring during differentiation. Transcription factor (TF) motif identification and correlation with TF binding site maps allowed the identification of TF circuitries acting on enhancers and SEs during differentiation. Overall, our study provides a broad, genome-wide description of chromatin dynamics and differential enhancer and promoter usage during epithelial differentiation, and describes a novel approach to identify active regulatory elements in rare stem cell populations.
Di Agostino S, Sorrentino G, Ingallina E, Valenti F, Ferraiuolo M, Bicciato S, Piazza S, Strano S, Del Sal G, Blandino G.
YAP enhances the pro-proliferative transcriptional activity of mutant p53 proteins.
EMBO Rep. 2016 Feb;17(2):188-201. doi: 10.15252/embr.201540488. Epub 2015 Dec 21. Abstract
Mutant p53 proteins are present in more than half of human cancers. Yes-associated protein (YAP) is a key transcriptional regulator controlling organ growth, tissue homeostasis, and cancer. Here, we report that these two determinants of human malignancy share common transcriptional signatures. YAP physically interacts with mutant p53 proteins in breast cancer cells and potentiates their pro-proliferative transcriptional activity. We found YAP as well as mutant p53 and the transcription factor NF-Y onto the regulatory regions of cyclin A, cyclin B, and CDK1 genes. Either mutant p53 or YAP depletion down-regulates cyclin A, cyclin B, and CDK1 gene expression and markedly slows the growth of diverse breast cancer cell lines. Pharmacologically induced cytoplasmic re-localization of YAP reduces the expression levels of cyclin A, cyclin B, and CDK1 genes both in vitro and in vivo. Interestingly, primary breast cancers carrying p53 mutations and displaying high YAP activity exhibit higher expression levels of cyclin A, cyclin B, and CDK1 genes when compared to wt-p53 tumors.
Salati S, Zini R, Nuzzo S, Guglielmelli P, Pennucci V, Prudente Z, Ruberti S, Rontauroli S, Norfo R, Bianchi E, Bogani C, Rotunno G, Fanelli T, Mannarelli C, Rosti V, Salmoiraghi S, Pietra D, Ferrari S, Barosi G, Rambaldi A, Cazzola M, Bicciato S, Tagliafico E, Vannucchi AM, Manfredini R; AGIMM (AIRC Gruppo Italiano Malattie Mieloproliferative) investigators.
Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in primary myelofibrosis.
Int J Cancer. 2016 Apr 1;138(7):1657-69. doi: 10.1002/ijc.29920. Epub 2015 Nov 25. Abstract
Primary myelofibrosis (PMF) is a Myeloproliferative Neoplasm (MPN) characterized by megakaryocyte hyperplasia, progressive bone marrow fibrosis, extramedullary hematopoiesis and transformation to Acute Myeloid Leukemia (AML). A number of phenotypic driver (JAK2, CALR, MPL) and additional subclonal mutations have been described in PMF, pointing to a complex genomic landscape. To discover novel genomic lesions that can contribute to disease phenotype and/or development, gene expression and copy number signals were integrated and several genomic abnormalities leading to a concordant alteration in gene expression levels were identified. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus was accompanied by a coordinated transcriptional up-regulation in PMF patients. PAOX inhibition resulted in rapid cell death of PMF progenitor cells, while sparing normal cells, suggesting that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells’ survival. Moreover, copy number loss in the chromatin modifier HMGXB4 gene correlates with a concomitant transcriptional down-regulation in PMF patients. Interestingly, silencing of HMGXB4 induces megakaryocyte differentiation, while inhibiting erythroid development, in human hematopoietic stem/progenitor cells. These results highlight a previously un-reported, yet potentially interesting role of HMGXB4 in the hematopoietic system and suggest that genomic and transcriptional imbalances of HMGXB4 could contribute to the aberrant expansion of the megakaryocytic lineage that characterizes PMF patients.
Volpi C, Mondanelli G, Pallotta MT, Vacca C, Iacono A, Gargaro M, Albini E, Bianchi R, Belladonna ML, Celanire S, Mordant C, Heroux M, Royer-Urios I, Schneider M, Vitte PA, Cacquevel M, Galibert L, Poli SM, Solari A, Bicciato S, Calvitti M, Antognelli C, Puccetti P, Orabona C, Fallarino F, Grohmann U.
Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells.
Neuropharmacology. 2016 Mar;102:59-71. doi: 10.1016/j.neuropharm.2015.10.036. Epub 2015 Oct 30. Abstract
Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild-yet chronic-neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1-but not pertussis toxin, which affects Gi protein-dependent responses-abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis.
Hudspeth K, Donadon M, Cimino M, Pontarini E, Tentorio P, Preti M, Hong M, Bertoletti A, Bicciato S, Invernizzi P, Lugli E, Torzilli G, Gershwin ME, Mavilio D.
Human liver-resident CD56(bright)/CD16(neg) NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways.
J Autoimmun. 2016 Jan;66:40-50. doi: 10.1016/j.jaut.2015.08.011. Epub 2015 Aug 30. Abstract
The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic.
We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts.
Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56(bright) lr-NK cells that localize within hepatic sinusoids. CD56(bright) lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3(pos) Kupffer cells, CXCL16(pos) endothelial cells and CCL5(pos) T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56(bright) NK cells that constitutively express CCR5 and CXCR6. CD56(bright) lr-NK cells co-exist with CD56(dim) conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56(dim) c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1.
Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56(bright) lr-NK cells will require modification of hepatic CCR5 and CXCR6.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Caroli J, Taccioli C, De La Fuente A, Serafini P, Bicciato S.
APTANI: a computational tool to select aptamers through sequence-structure motif analysis of HT-SELEX data.
Bioinformatics. 2016 Jan 15;32(2):161-4. doi: 10.1093/bioinformatics/btv545. Epub 2015 Sep 22. Abstract
Aptamers are synthetic nucleic acid molecules that can bind biological targets in virtue of both their sequence and three-dimensional structure. Aptamers are selected using SELEX, Systematic Evolution of Ligands by EXponential enrichment, a technique that exploits aptamer-target binding affinity. The SELEX procedure, coupled with high-throughput sequencing (HT-SELEX), creates billions of random sequences capable of binding different epitopes on specific targets. Since this technique produces enormous amounts of data, computational analysis represents a critical step to screen and select the most biologically relevant sequences.
Here, we present APTANI, a computational tool to identify target-specific aptamers from HT-SELEX data and secondary structure information. APTANI builds on AptaMotif algorithm, originally implemented to analyze SELEX data; extends the applicability of AptaMotif to HT-SELEX data and introduces new functionalities, as the possibility to identify binding motifs, to cluster aptamer families or to compare output results from different HT-SELEX cycles. Tabular and graphical representations facilitate the downstream biological interpretation of results.
AVAILABILITY AND IMPLEMENTATION:
APTANI is available at http://aptani.unimore.it.
Dyar KA, Ciciliot S, Tagliazucchi GM, Pallafacchina G, Tothova J, Argentini C, Agatea L, Abraham R, Ahdesmäki M, Forcato M, Bicciato S, Schiaffino S, Blaauw B.
The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle.
Mol Metab. 2015 Sep 25;4(11):823-33. doi: 10.1016/j.molmet.2015.09.004. eCollection 2015 Nov. Abstract
Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle.
We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression.
We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes.
We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca(2+)-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.
Taccioli C, Sorrentino G, Zannini A, Caroli J, Beneventano D, Anderlucci L, Lolli M, Bicciato S, Del Sal G.
MDP, a database linking drug response data to genomic information, identifies dasatinib and statins as a combinatorial strategy to inhibit YAP/TAZ in cancer cells.
Oncotarget. 2015 Nov 17;6(36):38854-65. doi: 10.18632/oncotarget.5749. Abstract
Targeted anticancer therapies represent the most effective pharmacological strategies in terms of clinical responses. In this context, genetic alteration of several oncogenes represents an optimal predictor of response to targeted therapy. Integration of large-scale molecular and pharmacological data from cancer cell lines promises to be effective in the discovery of new genetic markers of drug sensitivity and of clinically relevant anticancer compounds. To define novel pharmacogenomic dependencies in cancer, we created the Mutations and Drugs Portal (MDP, http://mdp.unimore.it), a web accessible database that combines the cell-based NCI60 screening of more than 50,000 compounds with genomic data extracted from the Cancer Cell Line Encyclopedia and the NCI60 DTP projects. MDP can be queried for drugs active in cancer cell lines carrying mutations in specific cancer genes or for genetic markers associated to sensitivity or resistance to a given compound. As proof of performance, we interrogated MDP to identify both known and novel pharmacogenomics associations and unveiled an unpredicted combination of two FDA-approved compounds, namely statins and Dasatinib, as an effective strategy to potently inhibit YAP/TAZ in cancer cells.
Zanconato F, Forcato M, Battilana G, Azzolin L, Quaranta E, Bodega B, Rosato A, Bicciato S, Cordenonsi M, Piccolo S.
Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth.
Nat Cell Biol. 2015 Sep;17(9):1218-27. doi: 10.1038/ncb3216. Epub 2015 Aug 10. Abstract
YAP/TAZ are nuclear effectors of the Hippo pathway regulating organ growth and tumorigenesis. Yet, their function as transcriptional regulators remains underinvestigated. By ChIP-seq analyses in breast cancer cells, we discovered that the YAP/TAZ transcriptional response is pervasively mediated by a dual element: TEAD factors, through which YAP/TAZ bind to DNA, co-occupying chromatin with activator protein-1 (AP-1, dimer of JUN and FOS proteins) at composite cis-regulatory elements harbouring both TEAD and AP-1 motifs. YAP/TAZ/TEAD and AP-1 form a complex that synergistically activates target genes directly involved in the control of S-phase entry and mitosis. This control occurs almost exclusively from distal enhancers that contact target promoters through chromatin looping. YAP/TAZ-induced oncogenic growth is strongly enhanced by gain of AP-1 and severely blunted by its loss. Conversely, AP-1-promoted skin tumorigenesis is prevented in YAP/TAZ conditional knockout mice. This work highlights a new layer of signalling integration, feeding on YAP/TAZ function at the chromatin level.
Guarneri V, Dieci MV, Frassoldati A, Maiorana A, Ficarra G, Bettelli S, Tagliafico E, Bicciato S, Generali DG, Cagossi K, Bisagni G, Sarti S, Musolino A, Ellis C, Crescenzo R, Conte P.
Prospective Biomarker Analysis of the Randomized CHER-LOB Study Evaluating the Dual Anti-HER2 Treatment With Trastuzumab and Lapatinib Plus Chemotherapy as Neoadjuvant Therapy for HER2-Positive Breast Cancer.
Oncologist. 2015 Sep;20(9):1001-10. doi: 10.1634/theoncologist.2015-0138. Epub 2015 Aug 5. Abstract
The CHER-LOB randomized phase II study showed that the combination of lapatinib and trastuzumab plus chemotherapy increases the pathologic complete remission (pCR) rate compared with chemotherapy plus either trastuzumab or lapatinib. A biomarker program was prospectively planned to identify potential predictors of sensitivity to different treatments and to evaluate treatment effect on tumor biomarkers.
MATERIALS AND METHODS:
Overall, 121 breast cancer patients positive for human epidermal growth factor 2 (HER2) were randomly assigned to neoadjuvant chemotherapy plus trastuzumab, lapatinib, or both trastuzumab and lapatinib. Pre- and post-treatment samples were centrally evaluated for HER2, p95-HER2, phosphorylated AKT (pAKT), phosphatase and tensin homolog, Ki67, apoptosis, and PIK3CA mutations. Fresh-frozen tissue samples were collected for genomic analyses.
A mutation in PIK3CA exon 20 or 9 was documented in 20% of cases. Overall, the pCR rates were similar in PIK3CA wild-type and PIK3CA-mutated patients (33.3% vs. 22.7%; p = .323). For patients receiving trastuzumab plus lapatinib, the probability of pCR was higher in PIK3CA wild-type tumors (48.4% vs. 12.5%; p = .06). Ki67, pAKT, and apoptosis measured on the residual disease were significantly reduced from baseline. The degree of Ki67 inhibition was significantly higher in patients receiving the dual anti-HER2 blockade. The integrated analysis of gene expression and copy number data demonstrated that a 50-gene signature specifically predicted the lapatinib-induced pCR.
PIK3CA mutations seem to identify patients who are less likely to benefit from dual anti-HER2 inhibition. p95-HER2 and markers of phosphoinositide 3-kinase pathway deregulation are not confirmed as markers of different sensitivity to trastuzumab or lapatinib.
IMPLICATIONS FOR PRACTICE:
HER2 is currently the only validated marker to select breast cancer patients for anti-HER2 treatment; however, it is becoming evident that HER2-positive breast cancer is a heterogeneous disease. In addition, more and more new anti-HER2 treatments are becoming available. There is a need to identify markers of sensitivity to different treatments to move in the direction of treatment personalization. This study identified PIK3CA mutations as a potential predictive marker of resistance to dual anti-HER2 treatment that should be further studied in breast cancer.
Rossi L, Forte D, Migliardi G, Salvestrini V, Buzzi M, Ricciardi MR, Licchetta R, Tafuri A, Bicciato S, Cavo M, Catani L, Lemoli RM, Curti A.
The tissue inhibitor of metalloproteinases 1 increases the clonogenic efficiency of human hematopoietic progenitor cells through CD63/PI3K/Akt signaling.
Exp Hematol. 2015 Nov;43(11):974-985.e1. doi: 10.1016/j.exphem.2015.07.003. Epub 2015 Jul 23. Abstract
Initially described as an endogenous inhibitor of proteases, the tissue inhibitor of metalloproteinases 1 (TIMP-1) also displays cytokine-like functions. TIMP-1 is a soluble protein whose levels are increased under inflammatory conditions. We recently found that TIMP-1(-/-) mice have decreased bone marrow (BM) cellularity and that the engraftment capability of TIMP-1(-/-) hematopoietic stem cells (HSCs) is impaired, owing to proliferation defects. Here, we investigated the role of recombinant human TIMP-1 (rhTIMP-1) in human hematopoietic stem/progenitor cells (HSPCs) and elucidated the downstream pathway ignited by rhTIMP-1. We found that rhTIMP-1 affects in vitro cell survival, proliferation, and particularly clonogenic expansion of CD34(+) HSPCs without compromising their short-term engraftment potential after transplantation into immunodeficient mice. These effects are independent on matrix metalloproteinase (MMP) inhibition and rely on TIMP-1’s binding to the tetraspanin membrane receptor CD63. Further investigation indicated that rhTIMP-1 stimulation induces phosphatidylinositol 3-kinase (PI3K) recruitment and Akt phosphorylation, both presiding over survival/proliferation pathways in HSPCs. Downstream targets of phosphorylated Akt (pAkt) are also modulated, including the proliferation marker cyclin D1 (CycD1), whose levels are increased upon exposure to rhTIMP-1. These findings indicate that rhTIMP-1 promotes clonogenic expansion and survival in human progenitors via the activation of the CD63/PI3K/pAkt signaling pathway, suggesting that TIMP-1 might be a key player in the network of proinflammatory factors modulating HSPC functions.
Grilli A, Sciandra M, Terracciano M, Picci P, Scotlandi K
Integrated approaches to miRNAs target definition: time-series analysis in an osteosarcoma differentiative model.
BMC Med Genomics. 2015 Jun 30;8:34. doi: 10.1186/s12920-015-0106-0. Abstract
microRNAs (miRs) are small non-coding RNAs involved in the fine regulation of several cellular processes by inhibiting their target genes at post-transcriptional level. Osteosarcoma (OS) is a tumor thought to be related to a molecular blockade of the normal process of osteoblast differentiation. The current paper explores temporal transcriptional modifications comparing an osteosarcoma cell line, Saos-2, and clones stably transfected with CD99, a molecule which was found to drive OS cells to terminally differentiate.
Parental cell line and CD99 transfectants were cultured up to 14 days in differentiating medium. In this setting, OS cells were profiled by gene and miRNA expression arrays. Integration of gene and miRNA profiling was performed by both sequence complementarity and expression correlation. Further enrichment and network analyses were carried out to focus on the modulated pathways and on the interactions between transcriptome and miRNome. To track the temporal transcriptional modification, a PCA analysis with differentiated human MSC was performed.
We identified a strong (about 80 %) gene down-modulation where reversion towards the osteoblast-like phenotype matches significant enrichment in TGFbeta signaling players like AKT1 and SMADs. In parallel, we observed the modulation of several cancer-related microRNAs like miR-34a, miR-26b or miR-378. To decipher their impact on the modified transcriptional program in CD99 cells, we correlated gene and microRNA time-series data miR-34a, in particular, was found to regulate a distinct subnetwork of genes with respect to the rest of the other differentially expressed miRs and it appeared to be the main mediator of several TGFbeta signaling genes at initial and middle phases of differentiation. Integration studies further highlighted the involvement of TGFbeta pathway in the differentiation of OS cells towards osteoblasts and its regulation by microRNAs.
These data underline that the expression of miR-34a and down-modulation of TGFbeta signaling emerge as pivotal events to drive CD99-mediated reversal of malignancy and activation of differentiation in OS cells. Our results describe crucial and specific interacting actors providing and supporting their relevance as potential targets for therapeutic differentiative strategies.
Poletti V, Delli Carri A, Malagoli Tagliazucchi G, Faedo A, Petiti L, Mazza EM, Peano C, De Bellis G, Bicciato S, Miccio A, Cattaneo E, Mavilio F.
Genome-Wide Definition of Promoter and Enhancer Usage during Neural Induction of Human Embryonic Stem Cells.
PLoS One. 2015 May 15;10(5):e0126590. doi: 10.1371/journal.pone.0126590. eCollection 2015. Abstract
Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a “bivalent” histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate.
Vermezovic J, Adamowicz M, Santarpia L, Rustighi A, Forcato M, Lucano C, Massimiliano L, Costanzo V, Bicciato S, Del Sal G, d’Adda di Fagagna F.
Notch is a direct negative regulator of the DNA-damage response.
Nat Struct Mol Biol. 2015 May;22(5):417-24. doi: 10.1038/nsmb.3013. Epub 2015 Apr 20. Abstract
The DNA-damage response (DDR) ensures genome stability and proper inheritance of genetic information, both of which are essential to survival. It is presently unclear to what extent other signaling pathways modulate DDR function. Here we show that Notch receptor binds and inactivates ATM kinase and that this mechanism is evolutionarily conserved in Caenorhabditis elegans, Xenopus laevis and humans. In C. elegans, the Notch pathway impairs DDR signaling in gonad germ cells. In mammalian cells, activation of human Notch1 leads to reduced ATM signaling in a manner independent of Notch1 transcriptional activity. Notch1 binds directly to the regulatory FATC domain of ATM and inhibits ATM kinase activity. Notch1 and ATM activation are inversely correlated in human breast cancers, and inactivation of ATM by Notch1 contributes to the survival of Notch1-driven leukemia cells upon DNA damage.
Enzo E, Santinon G, Pocaterra A, Aragona M, Bresolin S, Forcato M, Grifoni D, Pession A, Zanconato F, Guzzo G, Bicciato S, Dupont S.
Aerobic glycolysis tunes YAP/TAZ transcriptional activity.
EMBO J. 2015 May 12;34(10):1349-70. doi: 10.15252/embj.201490379. Epub 2015 Mar 21. Abstract
Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro-tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth-promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro-tumorigenic activities such as YAP/TAZ.
Cieri N, Oliveira G, Greco R, Forcato M, Taccioli C, Cianciotti B, Valtolina V, Noviello M, Vago L, Bondanza A, Lunghi F, Marktel S, Bellio L, Bordignon C, Bicciato S, Peccatori J, Ciceri F, Bonini C.
Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation.
Blood. 2015 Apr 30;125(18):2865-74. doi: 10.1182/blood-2014-11-608539. Epub 2015 Mar 3. Abstract
Memory stem T cells (TSCM) have been proposed as key determinants of immunologic memory. However, their exact contribution to a mounting immune response, as well as the mechanisms and timing of their in vivo generation, are poorly understood. We longitudinally tracked TSCM dynamics in patients undergoing haploidentical hematopoietic stem cell transplantation (HSCT), thereby providing novel hints on the contribution of this subset to posttransplant immune reconstitution in humans. We found that donor-derived TSCM are highly enriched early after HSCT. We showed at the antigen-specific and clonal level that TSCM lymphocytes can differentiate directly from naive precursors infused within the graft and that the extent of TSCM generation might correlate with interleukin 7 serum levels. In vivo fate mapping through T-cell receptor sequencing allowed defining the in vivo differentiation landscapes of human naive T cells, supporting the notion that progenies of single naive cells embrace disparate fates in vivo and highlighting TSCM as relevant novel players in the diversification of immunological memory after allogeneic HSCT.
Mazza EM, Zoso A, Mandruzzato S, Bronte V, Serafini P, Inverardi L, Bicciato S.
Gene expression profiling of human fibrocytic myeloid-derived suppressor cells (f-MDSCs).
Genom Data. 2014 Oct 31;2:389-92. doi: 10.1016/j.gdata.2014.10.018. eCollection 2014 Dec. Abstract
Myeloid-derived suppressor cells (MDSCs) have been shown to control self-reactive and anti-graft effector T-cells in autoimmunity and transplantation, but their therapeutic use is limited by their scarce availability in the peripheral blood of tumor-free donors. We isolated and characterized a novel population of myeloid suppressor cells, named fibrocytic MDSC (f-MDSC), which are differentiated from umbilical cord blood (UCB) precursors (Zoso et al., 2014). This MDSC subset promotes regulatory T-cell expansion and induces normoglycemia in a xenogeneic model of type 1 diabetes. Here we describe in details the experimental design and the bioinformatics analyses of the gene expression dataset used to investigate the molecular mechanisms at the base of MDSC tolerogenic and suppressive properties. We also provide an R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset. Raw and pre-processed data are available at Gene Expression Omnibus under accession GSE52376.
Di Minin G, Bellazzo A, Dal Ferro M, Chiaruttini G, Nuzzo S, Bicciato S, Piazza S, Rami D, Bulla R, Sommaggio R, Rosato A, Del Sal G, Collavin L.
Mutant p53 reprograms TNF signaling in cancer cells through interaction with the tumor suppressor DAB2IP.
Mol Cell. 2014 Dec 4;56(5):617-29. doi: 10.1016/j.molcel.2014.10.013. Epub 2014 Nov 13. Abstract
Inflammation is a significant factor in cancer development, and a molecular understanding of the parameters dictating the impact of inflammation on cancers could significantly improve treatment. The tumor suppressor p53 is frequently mutated in cancer, and p53 missense mutants (mutp53) can acquire oncogenic properties. We report that cancer cells with mutp53 respond to inflammatory cytokines increasing their invasive behavior. Notably, this action is coupled to expression of chemokines that can expose the tumor to host immunity, potentially affecting response to therapy. Mechanistically, mutp53 fuels NF-κB activation while it dampens activation of ASK1/JNK by TNFα, and this action depends on mutp53 binding and inhibiting the tumor suppressor DAB2IP in the cytoplasm. Interfering with such interaction reduced aggressiveness of cancer cells in xenografts. This interaction is an unexplored mechanism by which mutant p53 can influence tumor evolution, with implications for our understanding of the complex role of inflammation in cancer.
Peano C, Damiano F, Forcato M, Pietrelli A, Palumbo C, Corti G, Siculella L, Fuligni F, Tagliazucchi GM, De Benedetto GE, Bicciato S, De Bellis G, Alifano P.
Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering.
Metab Eng. 2014 Nov;26:1-16. doi: 10.1016/j.ymben.2014.08.001. Epub 2014 Aug 19. Abstract
Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer.
Marino MT, Grilli A, Baricordi C, Manara MC, Ventura S, Pinca RS, Bellenghi M, Calvaruso M, Mattia G, Donati D, Tripodo C, Picci P, Ferrari S, Scotlandi K.
Prognostic significance of miR-34a in Ewing sarcoma is associated with cyclin D1 and ki-67 expression.
Ann Oncol. 2014 Oct;25(10):2080-6. doi: 10.1093/annonc/mdu249. Epub 2014 Jul 10. Abstract
At diagnosis, identification of reliable biological indicators of prognosis to allow stratification of patients according to different risks is an important but still unresolved aspect in the treatment of Ewing sarcoma (EWS) patients. This study aimed to explore the role of miR-34A expression on prognosis of EWS patients.
PATIENTS AND METHODS:
Specimens from 109 patients with non-metastatic EWS treated at the Rizzoli Institute with neoadjuvant chemotherapy (protocols ISG/SSGIII, EW-1, EW-2, EW-REN2, EW-REN3, EW-PILOT) and 17 metastases were studied. Sixty-eight patients (62%) remained disease-free and 41 (38%) relapsed (median follow-up: 67 months, range 9-241 months). Expression of miR-34a and of some of its targets (cyclin D1, bcl-2, SIRT1 and YY1) was evaluated by qRT-PCR using TaqMan MicroRNA Assays and/or by immunohistochemistry on tissue microarrays from the same patients.
High expression of miR-34a in localized tumors was significantly related to better event-free and overall survival (P = 0.004). Relevance of miR-34a was confirmed by using different calibrators (normal mesenchymal stem cells and different normal tissues). By multivariate Cox regression analysis, low miR-34a expression as well as nontotal necrosis and high levels of lactate dehydrogenase were all confirmed as independent risk factors associated with poor outcome. Expression of miR-34a was lower in metastases than in primary tumors. It inversely correlated with expression of cyclin D1 and Ki-67.
By demonstrating its relationship with clinical outcome, we propose evaluation of miR-34a at diagnosis of EWS patients to allow early risk stratification. Validation of these results would nonetheless ultimately need a prospective assessment.
Zoso A, Mazza EM, Bicciato S, Mandruzzato S, Bronte V, Serafini P, Inverardi L.
Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion.
Eur J Immunol. 2014 Nov;44(11):3307-19. doi: 10.1002/eji.201444522. Epub 2014 Oct 18. Abstract
By restraining T-cell activation and promoting Treg-cell expansion, myeloid-derived suppressor cells (MDSCs) and tolerogenic DCs can control self-reactive and antigraft effector T cells in autoimmunity and transplantation. Their therapeutic use and characterization, however, is limited by their scarce availability in the peripheral blood of tumor-free donors. In the present study, we describe and characterize a novel population of human myeloid suppressor cells, named fibrocytic MDSC, which are differentiated from umbilical cord blood precursors by 4-day culture with FDA-approved cytokines (recombinant human-GM-CSF and recombinant human-G-CSF). This MDSC subset, characterized by the expression of MDSC-, DC-, and fibrocyte-associated markers, promotes Treg-cell expansion and induces normoglycemia in a xenogeneic mouse model of Type 1 diabetes. In order to exert their protolerogenic function, fibrocytic MDSCs require direct contact with activated T cells, which leads to the production and secretion of IDO. This new myeloid subset may have an important role in the in vitro and in vivo production of Treg cells for the treatment of autoimmune diseases, and in either the prevention or control of allograft rejection.
Riccardo F, Arigoni M, Buson G, Zago E, Iezzi M, Longo D, Carrara M, Fiore A, Nuzzo S, Bicciato S, Nanni P, Landuzzi L, Cavallo F, Calogero R, Quaglino E.
Characterization of a genetic mouse model of lung cancer: a promise to identify Non-Small Cell Lung Cancer therapeutic targets and biomarkers.
BMC Genomics. 2014;15 Suppl 3:S1. doi: 10.1186/1471-2164-15-S3-S1. Epub 2014 May 6. Abstract
Non-small cell lung cancer (NSCLC) accounts for 81% of all cases of lung cancer and they are often fatal because 60% of the patients are diagnosed at an advanced stage. Besides the need for earlier diagnosis, there is a high need for additional effective therapies. In this work, we investigated the feasibility of a lung cancer progression mouse model, mimicking features of human aggressive NSCLC, as biological reservoir for potential therapeutic targets and biomarkers.
We performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-ras(LA1)/p53(R172HΔg) and wild type (WT) mice to detect fusion genes and gene/exon-level differential expression associated to the increase of tumor mass. Fusion events were not detected in K-ras(LA1)/p53(R172HΔg) tumors. Differential expression at exon-level detected 33 genes with differential exon usage. Among them nine, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of more than 500 NSCLC RNA-seq transcriptomes. None of the genes showed a significant correlation between exon-level expression and disease prognosis. Differential expression at gene-level allowed the identification of 1513 genes with a significant increase in expression associated to tumor mass increase. 74 genes, i.e. those secreted or expressed on the plasma membrane, were used for a meta-analysis of two transcriptomics datasets of human NSCLC samples, encompassing more than 900 samples. SPP1 was the only molecule whose over-expression resulted statistically related to poor outcome regarding both survival and metastasis formation. Two other molecules showed over-expression associated to poor outcome due to metastasis formation: GM-CSF and ADORA3. GM-CSF is a secreted protein, and we confirmed its expression in the supernatant of a cell line derived by a K-ras(LA1)/p53(R172HΔg) mouse tumor. ADORA3 is instead involved in the induction of p53-mediated apoptosis in lung cancer cell lines. Since in our model p53 is inactivated, ADORA3 does not negatively affect tumor growth but remains expressed on tumor cells. Thus, it could represent an interesting target for the development of antibody-targeted therapy on a subset of NSCLC, which are p53 null and ADORA3 positive.
Our study provided a complete transcription overview of the K-ras(LA1)/p53(R172HΔg) mouse NSCLC model. This approach allowed the detection of ADORA3 as a potential target for antibody-based therapy in p53 mutated tumors.
Azzolin L, Panciera T, Soligo S, Enzo E, Bicciato S, Dupont S, Bresolin S, Frasson C, Basso G, Guzzardo V, Fassina A, Cordenonsi M, Piccolo S.
YAP/TAZ incorporation in the β-catenin destruction complex orchestrates the Wnt response.
Cell. 2014 Jul 3;158(1):157-70. doi: 10.1016/j.cell.2014.06.013. Epub 2014 Jun 26. Abstract
The Hippo transducers YAP/TAZ have been shown to play positive, as well as negative, roles in Wnt signaling, but the underlying mechanisms remain unclear. Here, we provide biochemical, functional, and genetic evidence that YAP and TAZ are integral components of the β-catenin destruction complex that serves as cytoplasmic sink for YAP/TAZ. In Wnt-ON cells, YAP/TAZ are physically dislodged from the destruction complex, allowing their nuclear accumulation and activation of Wnt/YAP/TAZ-dependent biological effects. YAP/TAZ are required for intestinal crypt overgrowth induced by APC deficiency and for crypt regeneration ex vivo. In Wnt-OFF cells, YAP/TAZ are essential for β-TrCP recruitment to the complex and β-catenin inactivation. In Wnt-ON cells, release of YAP/TAZ from the complex is instrumental for Wnt/β-catenin signaling. In line, the β-catenin-dependent maintenance of ES cells in an undifferentiated state is sustained by loss of YAP/TAZ. This work reveals an unprecedented signaling framework relevant for organ size control, regeneration, and tumor suppression.
Lomonaco V, Martoglia R, Mandreoli F, Anderlucci L, Emmett W, Bicciato S, Taccioli C.
UCbase 2.0: ultraconserved sequences database (2014 update).
Database (Oxford). 2014 Jun 19;2014. pii: bau062. doi: 10.1093/database/bau062. Print 2014. Abstract
UCbase 2.0 (http://ucbase.unimore.it) is an update, extension and evolution of UCbase, a Web tool dedicated to the analysis of ultraconserved sequences (UCRs). UCRs are 481 sequences >200 bases sharing 100% identity among human, mouse and rat genomes. They are frequently located in genomic regions known to be involved in cancer or differentially expressed in human leukemias and carcinomas. UCbase 2.0 is a platform-independent Web resource that includes the updated version of the human genome annotation (hg19), information linking disorders to chromosomal coordinates based on the Systematized Nomenclature of Medicine classification, a query tool to search for Single Nucleotide Polymorphisms (SNPs) and a new text box to directly interrogate the database using a MySQL interface. To facilitate the interactive visual interpretation of UCR chromosomal positioning, UCbase 2.0 now includes a graph visualization interface directly linked to UCSC genome browser. Database URL: http://ucbase.unimore.it.
Bessede A, Gargaro M, Pallotta MT, Matino D, Servillo G, Brunacci C, Bicciato S, Mazza EM, Macchiarulo A, Vacca C, Iannitti R, Tissi L, Volpi C, Belladonna ML, Orabona C, Bianchi R, Lanz TV, Platten M, Della Fazia MA, Piobbico D, Zelante T, Funakoshi H, Nakamura T, Gilot D, Denison MS, Guillemin GJ, DuHadaway JB, Prendergast GC, Metz R, Geffard M, Boon L, Pirro M, Iorio A, Veyret B, Romani L, Grohmann U, Fallarino F, Puccetti P.
Aryl hydrocarbon receptor control of a disease tolerance defence pathway.
Nature. 2014 Jul 10;511(7508):184-90. doi: 10.1038/nature13323. Abstract
Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.
Solieri L, Dakal TC, Bicciato S.
Quantitative phenotypic analysis of multistress response in Zygosaccharomyces rouxii complex.
FEMS Yeast Res. 2014 Jun;14(4):586-600. doi: 10.1111/1567-1364.12146. Epub 2014 Mar 13. Abstract
Zygosaccharomyces rouxii complex comprises three yeasts clusters sourced from sugar- and salt-rich environments: haploid Zygosaccharomyces rouxii, diploid Zygosaccharomyces sapae and allodiploid/aneuploid strains of uncertain taxonomic affiliations. These yeasts have been characterized with respect to gene copy number variation, karyotype variability and change in ploidy, but functional diversity in stress responses has not been explored yet. Here, we quantitatively analysed the stress response variation in seven strains of the Z. rouxii complex by modelling growth variables via model and model-free fitting methods. Based on the spline fit as most reliable modelling method, we resolved different interstrain responses to 15 environmental perturbations. Compared with Z. rouxii CBS 732(T) and Z. sapae strains ABT301(T) and ABT601, allodiploid strain ATCC 42981 and aneuploid strains CBS 4837 and CBS 4838 displayed higher multistress resistance and better performance in glycerol respiration even in the presence of copper. μ-based logarithmic phenotypic index highlighted that ABT601 is a slow-growing strain insensitive to stress, whereas ABT301(T) grows fast on rich medium and is sensitive to suboptimal conditions. Overall, the differences in stress response could imply different adaptation mechanisms to sugar- and salt-rich niches. The obtained phenotypic profiling contributes to provide quantitative insights for elucidating the adaptive mechanisms to stress in halo- and osmo-tolerant Zygosaccharomyces yeasts.
Italiani P, Mazza EM, Lucchesi D, Cifola I, Gemelli C, Grande A, Battaglia C, Bicciato S, Boraschi D.
Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro.
PLoS One. 2014 Feb 3;9(2):e87680. doi: 10.1371/journal.pone.0087680. eCollection 2014. Abstract
Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangements.
Gemelli C, Zanocco Marani T, Bicciato S, Mazza EM, Boraschi D, Salsi V, Zappavigna V, Parenti S, Selmi T, Tagliafico E, Ferrari S, Grande A.
MafB is a downstream target of the IL-10/STAT3 signaling pathway, involved in the regulation of macrophage de-activation.
Biochim Biophys Acta. 2014 May;1843(5):955-64. doi: 10.1016/j.bbamcr.2014.01.021. Epub 2014 Jan 25. Abstract
In spite of the numerous reports implicating MafB transcription factor in the molecular control of monocyte-macrophage differentiation, the precise genetic program underlying this activity has been, to date, poorly understood. To clarify this issue, we planned a number of experiments that were mainly conducted on human primary macrophages. In this regard, a preliminary gene function study, based on MafB inactivation and over-expression, indicated MMP9 and IL-7R genes as possible targets of the investigated transcription factor. Bioinformatics analysis of their promoter regions disclosed the presence of several putative MARE elements and a combined approach of EMSA and luciferase assay subsequently demonstrated that expression of both genes is indeed activated by MafB through a direct transcription mechanism. Additional investigation, performed with similar procedures to elucidate the biological relevance of our observation, revealed that MafB is a downstream target of the IL-10/STAT3 signaling pathway, normally inducing the macrophage de-activation process. Taken together our data support the existence of a signaling cascade by which stimulation of macrophages with the IL-10 cytokine determines a sequential activation of STAT3 and MafB transcription factors, in turn leading to an up-regulated expression of MMP9 and IL-7R genes.
Rustighi A, Zannini A, Tiberi L, Sommaggio R, Piazza S, Sorrentino G, Nuzzo S, Tuscano A, Eterno V, Benvenuti F, Santarpia L, Aifantis I, Rosato A, Bicciato S, Zambelli A, Del Sal G.
Prolyl-isomerase Pin1 controls normal and cancer stem cells of the breast.
EMBO Mol Med. 2014 Jan;6(1):99-119. doi: 10.1002/emmm.201302909. Epub 2013 Dec 15. Erratum in: EMBO Mol Med. 2014 May;6(5):702. Abstract
Mammary epithelial stem cells are fundamental to maintain tissue integrity. Cancer stem cells (CSCs) are implicated in both treatment resistance and disease relapse, and the molecular bases of their malignant properties are still poorly understood. Here we show that both normal stem cells and CSCs of the breast are controlled by the prolyl-isomerase Pin1. Mechanistically, following interaction with Pin1, Notch1 and Notch4, key regulators of cell fate, escape from proteasomal degradation by their major ubiquitin-ligase Fbxw7α. Functionally, we show that Fbxw7α acts as an essential negative regulator of breast CSCs’ expansion by restraining Notch activity, but the establishment of a Notch/Pin1 active circuitry opposes this effect, thus promoting breast CSCs self-renewal, tumor growth and metastasis in vivo. In human breast cancers, despite Fbxw7α expression, high levels of Pin1 sustain Notch signaling, which correlates with poor prognosis. Suppression of Pin1 holds promise in reverting aggressive phenotypes, through CSC exhaustion as well as recovered drug sensitivity carrying relevant implications for therapy of breast cancers.
Dyar KA, Ciciliot S, Wright LE, Biensø RS, Tagliazucchi GM, Patel VR, Forcato M, Paz MI, Gudiksen A, Solagna F, Albiero M, Moretti I, Eckel-Mahan KL, Baldi P, Sassone-Corsi P, Rizzuto R, Bicciato S, Pilegaard H, Blaauw B, Schiaffino S.
Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock.
Mol Metab. 2013 Oct 23;3(1):29-41. doi: 10.1016/j.molmet.2013.10.005. eCollection 2014 Feb. Erratum in: Mol Metab. 2014 Dec;3(9):857. Abstract
Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.
Cieri N, Camisa B, Cocchiarella F, Forcato M, Oliveira G, Provasi E, Bondanza A, Bordignon C, Peccatori J, Ciceri F, Lupo-Stanghellini MT, Mavilio F, Mondino A, Bicciato S, Recchia A, Bonini C.
IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors.
Blood. 2013 Jan 24;121(4):573-84. doi: 10.1182/blood-2012-05-431718. Epub 2012 Nov 15. Abstract
Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.
Moreno-Asso A, Castaño C, Grilli A, Novials A, Servitja JM.
Glucose regulation of a cell cycle gene module is selectively lost in mouse pancreatic islets during ageing.
Diabetologia. 2013 Aug;56(8):1761-72. doi: 10.1007/s00125-013-2930-0. Epub 2013 May 18. Abstract
Transcriptional networks in beta cells are modulated by extracellular signals such as glucose, thereby ensuring beta cell adaptation to systemic insulin demands. Ageing is a main risk factor for type 2 diabetes and has been associated with perturbed expression of genes essential for beta cell function. We aimed to uncover glucose-dependent gene modules in mouse pancreatic islets and investigate how this regulation is affected by ageing.
Global gene expression was assessed in pancreatic islets from young and aged wild-type and Cdkn2a (Ink4a/Arf)-deficient mice exposed to different glucose concentrations. Gene modules were identified by gene ontology and gene set enrichment analysis.
Gene expression profiling revealed that variations in glucose levels have a widespread and highly dynamic impact on the islet transcriptome. Stimulatory glucose levels induced the expression of highly beta cell-selective genes and repressed the expression of ubiquitous genes involved in stress and antiproliferative responses, and in organelle biogenesis. Interestingly, a module comprising cell cycle genes was significantly induced between non-stimulatory and stimulatory glucose concentrations. Unexpectedly, glucose regulation of gene expression was broadly maintained in islets from old mice. However, glucose induction of mitotic genes was selectively lost in aged islets and was not even restored in the absence of the cell cycle inhibitors p16(INK4a) and p19(ARF), which have been implicated in the restricted proliferative capacity of beta cells with advanced age.
Glucose-dependent transcriptional networks in islets are globally conserved during ageing, with the exception of the ability of stimulatory glucose levels to induce a cell cycle gene module.
Lahti L, Schäfer M, Klein HU, Bicciato S, Dugas M.
Cancer gene prioritization by integrative analysis of mRNA expression and DNA copy number data: a comparative review.
Brief Bioinform. 2013 Jan;14(1):27-35. doi: 10.1093/bib/bbs005. Epub 2012 Mar 22. Review. Abstract
A variety of genome-wide profiling techniques are available to investigate complementary aspects of genome structure and function. Integrative analysis of heterogeneous data sources can reveal higher level interactions that cannot be detected based on individual observations. A standard integration task in cancer studies is to identify altered genomic regions that induce changes in the expression of the associated genes based on joint analysis of genome-wide gene expression and copy number profiling measurements. In this review, we highlight common approaches to genomic data integration and provide a transparent benchmarking procedure to quantitatively compare method performances in cancer gene prioritization. Algorithms, data sets and benchmarking results are available at http://intcomp.r-forge.r-project.org.
Azzolin L, Zanconato F, Bresolin S, Forcato M, Basso G, Bicciato S, Cordenonsi M, Piccolo S.
Role of TAZ as mediator of Wnt signaling.
Cell. 2012 Dec 21;151(7):1443-56. doi: 10.1016/j.cell.2012.11.027. Epub 2012 Dec 13 Abstract
Wnt growth factors are fundamental regulators of cell fate, but how the Wnt signal is translated into biological responses is incompletely understood. Here, we report that TAZ, a biologically potent transcriptional coactivator, serves as a downstream element of the Wnt/β-catenin cascade. This function of TAZ is independent from its well-established role as mediator of Hippo signaling. In the absence of Wnt activity, the components of the β-catenin destruction complex–APC, Axin, and GSK3–are also required to keep TAZ at low levels. TAZ degradation depends on phosphorylated β-catenin that bridges TAZ to its ubiquitin ligase β-TrCP. Upon Wnt signaling, escape of β-catenin from the destruction complex impairs TAZ degradation and leads to concomitant accumulation of β-catenin and TAZ. At the genome-wide level, a substantial portion of Wnt transcriptional responses is mediated by TAZ. TAZ activation is a general feature of Wnt signaling and is functionally relevant to mediate Wnt biological effects.
Garofalo C, Mancarella C, Grilli A, Manara MC, Astolfi A, Marino MT, Conte A, Sigismund S, Carè A, Belfiore A, Picci P, Scotlandi K.
Identification of common and distinctive mechanisms of resistance to different anti-IGF-IR agents in Ewing’s sarcoma.
Mol Endocrinol. 2012 Sep;26(9):1603-16. doi: 10.1210/me.2012-1142. Epub 2012 Jul 13. Abstract
IGF system contributes significantly to many human malignancies. Targeting IGF-I receptor (IGF-IR) has been reported to be active against several tumors, but particular efficacy was observed only against a minority of Ewing’s sarcoma patients. Identification of mechanisms of acquired resistance to anti-IGF-IR agents is mandatory to individualize their use in clinics and optimize cure costs. In this study, we compared gene expression profiles of cells made resistant with three different anti-IGF-IR drugs (human antibodies AVE1642, Figitumumab, or tyrosine kinase inhibitor NVP-AEW541) to highlight common and distinctive mechanisms of resistance. Among common mechanisms, we identified two molecular signatures that distinguish sensitive from resistant cells. Annotation analysis indicated some common altered pathways, such as insulin signaling, MAPK pathway, endocytosis, and modulation of some members of the interferon-induced transmembrane protein family. Among distinctive pathways/processes, resistance to human antibodies involves mainly genes regulating neural differentiation and angiogenesis, whereas resistance to NVP-AEW541 is mainly associated with alterations in genes concerning inflammation and antigen presentation. Evaluation of the common altered pathways indicated that resistant cells seem to maintain intact the IGF-IR internalization/degradation route of sensitive cells but constantly down-regulated its expression. In resistant cells, the loss of proliferative stimulus, normally sustained by IGF-I/IGF-IR autocrine loop in Ewing’s sarcoma cells, is compensated by transcriptional up-regulation of IGF-II and insulin receptor-A; this signaling seems to favor the MAPK pathway over the v-akt murine thymoma viral oncogene homolog 1 pathway. Overall, complexity of IGF system requires analytical evaluation of its components to select those patients that may really benefit from this targeted therapy and support the idea of cotargeting IGF-IR and insulin receptor-A to increase the efficacy.
Maciotta S, Meregalli M, Cassinelli L, Parolini D, Farini A, Fraro GD, Gandolfi F, Forcato M, Ferrari S, Gabellini D, Bicciato S, Cossu G, Torrente Y.
Hmgb3 is regulated by microRNA-206 during muscle regeneration.
PLoS One. 2012;7(8):e43464. doi: 10.1371/journal.pone.0043464. Epub 2012 Aug 17. Abstract
MicroRNAs (miRNAs) have been recently involved in most of human diseases as targets for potential strategies to rescue the pathological phenotype. Since the skeletal muscle is a spread-wide highly differentiated and organized tissue, rescue of severely compromised muscle still remains distant from nowadays. For this reason, we aimed to identify a subset of miRNAs major involved in muscle remodelling and regeneration by analysing the miRNA-profile of single fibres isolated from dystrophic muscle, which was here considered as a model of chronic damage.
The miRNA-signature associated to regenerating (newly formed) and remodelling (resting) fibres was investigated in animal models of muscular dystrophies and acute damage, in order to distinguish which miRNAs are primary related to muscle regeneration. In this study we identify fourteen miRNAs associated to dystrophic fibres responsible for muscle regeneration and remodelling, and confirm over-expression of the previously identified regeneration-associated myomiR-206. In particular, a functional binding site for myomiR-206 was identified and validated in the 3’untranslated region (3’UTR) of an X-linked member of a family of sequence independent chromatin-binding proteins (Hmgb3) that is preferentially expressed in hematopoietic stem cells. During regeneration of single muscle fibres, Hmgb3 messenger RNA (mRNA) and protein expression was gradually reduced, concurrent with the up-regulation of miR-206.
Our results elucidate a negative feedback circuit in which myomiR-206 represses Hmgb3 expression to modulate the regeneration of single muscle fibres after acute and chronic muscle damage. These findings suggest that myomiR-206 may be a potential therapeutic target in muscle diseases.
Montagner M, Enzo E, Forcato M, Zanconato F, Parenti A, Rampazzo E, Basso G, Leo G, Rosato A, Bicciato S, Cordenonsi M, Piccolo S.
SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors.
Nature. 2012 Jul 19;487(7407):380-4. doi: 10.1038/nature11207. Abstract
The molecular determinants of malignant cell behaviours in breast cancer remain only partially understood. Here we show that SHARP1 (also known as BHLHE41 or DEC2) is a crucial regulator of the invasive and metastatic phenotype in triple-negative breast cancer (TNBC), one of the most aggressive types of breast cancer. SHARP1 is regulated by the p63 metastasis suppressor and inhibits TNBC aggressiveness through inhibition of hypoxia-inducible factor 1α (HIF-1α) and HIF-2α (HIFs). SHARP1 opposes HIF-dependent TNBC cell migration in vitro, and invasive or metastatic behaviours in vivo. SHARP1 is required, and sufficient, to limit expression of HIF-target genes. In primary TNBC, endogenous SHARP1 levels are inversely correlated with those of HIF targets. Mechanistically, SHARP1 binds to HIFs and promotes HIF proteasomal degradation by serving as the HIF-presenting factor to the proteasome. This process is independent of pVHL (von Hippel-Lindau tumour suppressor), hypoxia and the ubiquitination machinery. SHARP1 therefore determines the intrinsic instability of HIF proteins to act in parallel to, and cooperate with, oxygen levels. This work sheds light on the mechanisms and pathways by which TNBC acquires invasiveness and metastatic propensity.
Nakatani F, Ferracin M, Manara MC, Ventura S, Del Monaco V, Ferrari S, Alberghini M, Grilli A, Knuutila S, Schaefer KL, Mattia G, Negrini M, Picci P, Serra M, Scotlandi K.
miR-34a predicts survival of Ewing’s sarcoma patients and directly influences cell chemo-sensitivity and malignancy.
J Pathol. 2012 Apr;226(5):796-805. doi: 10.1002/path.3007. Abstract
Identification of factors to detect chemotherapy-resistant tumours at diagnosis is a first priority for risk-adapted therapy in the oncology of children and young adults, where more individualized, effective, and less toxic treatments are highly desirable. In this study, we analysed the miRNAs discriminating Ewing’s sarcoma (EWS) patients with different clinical outcomes in order to identify new indicators of prognosis. miRNA expression was investigated in 49 primary EWSs by using the Agilent human miRNA microarray v.2 and/or qRT-PCR. Statistical power of the samples studied for miRNA expression was verified, indicating adequate sample size. Microarray analysis defined a signature of five miRNAs (miR-34a, miR-23a, miR-92a, miR-490-3p, and miR-130b) as an independent predictor of risk for disease progression and survival. Validation analysis in the extended sample set indicated that both miR-34a and miR-490-3p achieved sufficient statistical power to predict prognosis. Results were particularly robust for miR-34a, which appeared associated with either event-free or overall survival and emerged as a significant predictor also after multivariate analysis. Patients with the highest expression of miR-34a did not experience adverse events in 5 years; in contrast, patients with the lowest expression recurred within 2 years. High expression of miR34a can be detected also in paraffin-embedded tissues by in situ hybridization, thus contributing to an easy routine evaluation of this miRNA. Functional analysis of miR-34a in EWS cell lines indicated that when miR-34a expression was enforced, cells were less proliferative, less malignant, and sensitized to doxorubicin and vincristine. Expression of miR-34a could be increased in p53wt cells by treatment with nutlin-3a. Accordingly, nutlin-3a synergizes with doxorubicin. Overall, our data indicate that miR-34a expression is a strong predictor of outcome in EWS. Restoration of miR-34a activity may be useful to decrease malignancy and increase tumour sensitivity to current drugs, so sparing excessive long-term toxicity to EWS patients.
Peano C, Talà A, Corti G, Pasanisi D, Durante M, Mita G, Bicciato S, De Bellis G, Alifano P.
Comparative genomics and transcriptional profiles of Saccharopolyspora erythraea NRRL 2338 and a classically improved erythromycin over-producing strain.
Microb Cell Fact. 2012 Mar 8;11:32. doi: 10.1186/1475-2859-11-32. Abstract
The molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood although this information is essential to design rational strategies for industrial strain improvement. In this study, we applied comparative genomics to identify all genetic changes occurring during the development of an erythromycin overproducer obtained using the traditional mutate-and- screen method.
Compared with the parental Saccharopolyspora erythraea NRRL 2338, the genome of the overproducing strain presents 117 deletion, 78 insertion and 12 transposition sites, with 71 insertion/deletion sites mapping within coding sequences (CDSs) and generating frame-shift mutations. Single nucleotide variations are present in 144 CDSs. Overall, the genomic variations affect 227 proteins of the overproducing strain and a considerable number of mutations alter genes of key enzymes in the central carbon and nitrogen metabolism and in the biosynthesis of secondary metabolites, resulting in the redirection of common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, affect global expression profile as demonstrated by a comparative analysis of the parental and overproducer expression profiles. Genomic data, finally, suggest that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes.
This study helps to clarify the mechanisms underlying antibiotic overproduction providing valuable information about new possible molecular targets for rationale strain improvement.
Cordenonsi M, Zanconato F, Azzolin L, Forcato M, Rosato A, Frasson C, Inui M, Montagner M, Parenti AR, Poletti A, Daidone MG, Dupont S, Basso G, Bicciato S, Piccolo S.
The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells.
Cell. 2011 Nov 11;147(4):759-72. doi: 10.1016/j.cell.2011.09.048. Abstract
Cancer stem cells (CSCs) are proposed to drive tumor initiation and progression. Yet, our understanding of the cellular and molecular mechanisms that underlie CSC properties is limited. Here we show that the activity of TAZ, a transducer of the Hippo pathway, is required to sustain self-renewal and tumor-initiation capacities in breast CSCs. TAZ protein levels and activity are elevated in prospective CSCs and in poorly differentiated human tumors and have prognostic value. Gain of TAZ endows self-renewal capacity to non-CSCs. In epithelial cells, TAZ forms a complex with the cell-polarity determinant Scribble, and loss of Scribble–or induction of the epithelial-mesenchymal transition (EMT)–disrupts the inhibitory association of TAZ with the core Hippo kinases MST and LATS. This study links the CSC concept to the Hippo pathway in breast cancer and reveals a mechanistic basis of the control of Hippo kinases by cell polarity.
Agnelli L, Forcato M, Ferrari F, Tuana G, Todoerti K, Walker BA, Morgan GJ, Lombardi L, Bicciato S, Neri A.
The reconstruction of transcriptional networks reveals critical genes with implications for clinical outcome of multiple myeloma.
Clin Cancer Res. 2011 Dec 1;17(23):7402-12. doi: 10.1158/1078-0432.CCR-11-0596. Epub 2011 Sep 2. Abstract
The combined use of microarray technologies and bioinformatics analysis has improved our understanding of biological complexity of multiple myeloma (MM). In contrast, the application of the same technology in the attempt to predict clinical outcome has been less successful with the identification of heterogeneous molecular signatures. Herein, we have reconstructed gene regulatory networks in a panel of 1,883 samples from MM patients derived from publicly available gene expression sets, to allow the identification of robust and reproducible signatures associated with poor prognosis across independent data sets.
Gene regulatory networks were reconstructed by using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) and microarray data from seven MM data sets. Critical analysis of network components was applied to identify genes playing an essential role in transcriptional networks, which are conserved between data sets.
Network critical analysis revealed that (i) CCND1 and CCND2 were the most critical genes; (ii) CCND2, AIF1, and BLNK had the largest number of connections shared among the data sets; (iii) robust gene signatures with prognostic power were derived from the most critical transcripts and from shared primary neighbors of the most connected nodes. Specifically, a critical-gene model, comprising FAM53B, KIF21B, WHSC1, and TMPO, and a neighbor-gene model, comprising BLNK shared neighbors CSGALNACT1 and SLC7A7, predicted survival in all data sets with follow-up information.
The reconstruction of gene regulatory networks in a large panel of MM tumors defined robust and reproducible signatures with prognostic importance, and may lead to identify novel molecular mechanisms central to MM biology.
Pallotta MT, Orabona C, Volpi C, Vacca C, Belladonna ML, Bianchi R, Servillo G, Brunacci C, Calvitti M, Bicciato S, Mazza EM, Boon L, Grassi F, Fioretti MC, Fallarino F, Puccetti P, Grohmann U.
Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells.
Nat Immunol. 2011 Jul 31;12(9):870-8. doi: 10.1038/ni.2077. Abstract
Regulation of tryptophan metabolism by indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs) is a highly versatile modulator of immunity. In inflammation, interferon-γ is the main inducer of IDO for the prevention of hyperinflammatory responses, yet IDO is also responsible for self-tolerance effects in the longer term. Here we show that treatment of mouse plasmacytoid DCs (pDCs) with transforming growth factor-β (TGF-β) conferred regulatory effects on IDO that were mechanistically separable from its enzymic activity. We found that IDO was involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in pDCs. Thus, IDO has a tonic, nonenzymic function that contributes to TGF-β-driven tolerance in noninflammatory contexts.
Ferrari F, Solari A, Battaglia C, Bicciato S.
PREDA: an R-package to identify regional variations in genomic data.
Bioinformatics. 2011 Sep 1;27(17):2446-7. doi: 10.1093/bioinformatics/btr404. Epub 2011 Jul 7. Abstract
Chromosomal patterns of genomic signals represent molecular fingerprints that may reveal how the local structural organization of a genome impacts the functional control mechanisms. Thus, the integrative analysis of multiple sources of genomic data and information deepens the resolution and enhances the interpretation of stand-alone high-throughput data. In this note, we present PREDA (Position RElated Data Analysis), an R package for detecting regional variations in genomics data. PREDA identifies relevant chromosomal patterns in high-throughput data using a smoothing approach that accounts for distance and density variability of genomics features. Custom-designed data structures allow efficiently managing diverse signals in different genomes. A variety of smoothing functions and statistics empower flexible and robust workflows. The modularity of package design allows an easy deployment of custom analytical pipelines. Tabular and graphical representations facilitate downstream biological interpretation of results.
Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S.
Role of YAP/TAZ in mechanotransduction.
Nature. 2011 Jun 8;474(7350):179-83. doi: 10.1038/nature10137. Abstract
Cells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues, such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translate these stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiation and cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nuclear transcription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) as nuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPase activity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival of endothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints in dictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by the cellular microenvironment.
Mosca L, Fabris S, Lionetti M, Todoerti K, Agnelli L, Morabito F, Cutrona G, Andronache A, Matis S, Ferrari F, Gentile M, Spriano M, Callea V, Festini G, Molica S, Deliliers GL, Bicciato S, Ferrarini M, Neri A.
Integrative genomics analyses reveal molecularly distinct subgroups of B-cell chronic lymphocytic leukemia patients with 13q14 deletion.
Clin Cancer Res. 2010 Dec 1;16(23):5641-53. doi: 10.1158/1078-0432.CCR-10-0151. Epub 2010 Oct 14. Abstract
Chromosome 13q14 deletion occurs in a substantial number of chronic lymphocytic leukemia (CLL) patients and it is believed to play a pathogenetic role. The exact mechanisms involved in this lesion have not yet been fully elucidated because of its heterogeneity and the imprecise knowledge of the implicated genes. This study was addressed to further contribute to the molecular definition of this lesion in CLL.
We applied single-nucleotide polymorphism (SNP)-array technology and gene expression profiling data to investigate the 13q14 deletion occurring in a panel of 100 untreated, early-stage (Binet A) patients representative of the major genetics, molecular, and biological features of the disease.
Concordantly with FISH analysis, SNP arrays identified 44 patients with del(13)(q14) including 11 cases with a biallelic deletion. The shorter monoallelic deletion was 635-kb long. The loss of the miR-15a/16-1 cluster occurred in all del(13)(q14) cases except in 2 patients with a monoallelic deletion, who retained both copies. MiR-15a/16 expression was significantly downregulated only in patients with the biallelic loss of the miRNA cluster compared to 13q normal cases. Finally, the natural grouping of SNP profiles by nonnegative matrix factorization algorithm showed that patients could be classified into 2 separate clusters, mainly characterized by short/biallelic versus wide/monoallelic 13q14 deletions. Supervised analyses of expression data showed that specific transcriptional profiles are correlated with these 2 genomic subgroups.
Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL.
Fallarino F, Volpi C, Fazio F, Notartomaso S, Vacca C, Busceti C, Bicciato S, Battaglia G, Bruno V, Puccetti P, Fioretti MC, Nicoletti F, Grohmann U, Di Marco R.
Metabotropic glutamate receptor-4 modulates adaptive immunity and restrains neuroinflammation.
Nat Med. 2010 Aug;16(8):897-902. doi: 10.1038/nm.2183. Epub 2010 Jul 25. Abstract
High amounts of glutamate are found in the brains of people with multiple sclerosis, an inflammatory disease marked by progressive demyelination. Glutamate might affect neuroinflammation via effects on immune cells. Knockout mice lacking metabotropic glutamate receptor-4 (mGluR4) were markedly vulnerable to experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis) and developed responses dominated by interleukin-17-producing T helper (T(H)17) cells. In dendritic cells (DCs) from those mice, defective mGluR4 signaling-which would normally decrease intracellular cAMP formation-biased T(H) cell commitment to the T(H)17 phenotype. In wild-type mice, mGluR4 was constitutively expressed in all peripheral DCs, and this expression increased after cell activation. Treatment of wild-type mice with a selective mGluR4 enhancer increased EAE resistance via regulatory T (T(reg)) cells. The high amounts of glutamate in neuroinflammation might reflect a counterregulatory mechanism that is protective in nature and might be harnessed therapeutically for restricting immunopathology in multiple sclerosis.
Marigo I, Bosio E, Solito S, Mesa C, Fernandez A, Dolcetti L, Ugel S, Sonda N, Bicciato S, Falisi E, Calabrese F, Basso G, Zanovello P, Cozzi E, Mandruzzato S, Bronte V.
Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor.
Immunity. 2010 Jun 25;32(6):790-802. doi: 10.1016/j.immuni.2010.05.010. Epub 2010 Jun 3. Abstract
Tumor growth is associated with a profound alteration in myelopoiesis, leading to recruitment of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). We showed that among factors produced by various experimental tumors, the cytokines GM-CSF, G-CSF, and IL-6 allowed a rapid generation of MDSCs from precursors present in mouse and human bone marrow (BM). BM-MDSCs induced by GM-CSF+IL-6 possessed the highest tolerogenic activity, as revealed by the ability to impair the priming of CD8(+) T cells and allow long term acceptance of pancreatic islet allografts. Cytokines inducing MDSCs acted on a common molecular pathway and the immunoregulatory activity of both tumor-induced and BM-derived MDSCs was entirely dependent on the C/EBPbeta transcription factor. Adoptive transfer of tumor antigen-specific CD8(+) T lymphocytes resulted in therapy of established tumors only in mice lacking C/EBPbeta in the myeloid compartment, suggesting that C/EBPbeta is a critical regulator of the immunosuppressive environment created by growing cancers.
Martello G, Rosato A, Ferrari F, Manfrin A, Cordenonsi M, Dupont S, Enzo E, Guzzardo V, Rondina M, Spruce T, Parenti AR, Daidone MG, Bicciato S, Piccolo S.
A MicroRNA targeting dicer for metastasis control.
Cell. 2010 Jun 25;141(7):1195-207. doi: 10.1016/j.cell.2010.05.017. Abstract
Although specific microRNAs (miRNAs) can be upregulated in cancer, global miRNA downregulation is a common trait of human malignancies. The mechanisms of this phenomenon and the advantages it affords remain poorly understood. Here we identify a microRNA family, miR-103/107, that attenuates miRNA biosynthesis by targeting Dicer, a key component of the miRNA processing machinery. In human breast cancer, high levels of miR-103/107 are associated with metastasis and poor outcome. Functionally, miR-103/107 confer migratory capacities in vitro and empower metastatic dissemination of otherwise nonaggressive cells in vivo. Inhibition of miR-103/107 opposes migration and metastasis of malignant cells. At the cellular level, a key event fostered by miR-103/107 is induction of epithelial-to-mesenchymal transition (EMT), attained by downregulating miR-200 levels. These findings suggest a new pathway by which Dicer inhibition drifts epithelial cancer toward a less-differentiated, mesenchymal fate to foster metastasis.
Ferracin M, Zagatti B, Rizzotto L, Cavazzini F, Veronese A, Ciccone M, Saccenti E, Lupini L, Grilli A, De Angeli C, Negrini M, Cuneo A.
MicroRNAs involvement in fludarabine refractory chronic lymphocytic leukemia.
Mol Cancer. 2010 May 26;9:123. doi: 10.1186/1476-4598-9-123. Abstract
Fludarabine, is one of the most active single agents in the treatment of chronic lymphocytic leukemia (CLL). Over time, however, virtually all CLL patients become fludarabine-refractory. To elucidate whether microRNAs are involved in the development of fludarabine resistance, we analyzed the expression of 723 human miRNAs before and 5-days after fludarabine mono-therapy in 17 CLL patients which were classified as responder or refractory to fludarabine treatment based on NCI criteria.
By comparing the expression profiles of these two groups of patients, we identified a microRNA signature able to distinguish refractory from sensitive CLLs. The expression of some microRNAs was also able to predict fludarabine resistance of 12 independent CLL patients. Among the identified microRNAs, miR-148a, miR-222 and miR-21 exhibited a significantly higher expression in non-responder patients either before and after fludarabine treatment. After performing messenger RNA expression profile of the same patients, the activation of p53-responsive genes was detected in fludarabine responsive cases only, therefore suggesting a possible mechanism linked to microRNA deregulation in non-responder patients. Importantly, inhibition of miR-21 and miR-222 by anti-miRNA oligonucleotides induced a significant increase in caspase activity in fludarabine-treated p53-mutant MEG-01 cells, suggesting that miR-21 and miR-222 up-regulation may be involved in the establishment of fludarabine resistance.
This is the first report that reveals the existence of a microRNA profile that differentiate refractory and sensitive CLLs, either before and after fludarabine mono-therapy. A p53 dysfunctional pathway emerged in refractory CLLs and could contribute in explaining the observed miRNA profile. Moreover, this work indicates that specific microRNAs can be used to predict fludarabine resistance and may potentially be used as therapeutic targets, therefore establishing an important starting point for future studies.
Sales G, Coppe A, Bicciato S, Bortoluzzi S, Romualdi C.
Impact of probe annotation on the integration of miRNA-mRNA expression profiles for miRNA target detection.
Nucleic Acids Res. 2010 Apr;38(7):e97. doi: 10.1093/nar/gkp1239. Epub 2010 Jan 13. Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional and translational levels by an imperfect binding to target mRNA 3’UTR regions. While the ab-initio computational prediction of miRNA-mRNA interactions still poses significant challenges, it is possible to overcome some of its limitations by carefully integrating into the analysis the paired expression profiles of miRNAs and mRNAs. In this work, we show how the choice of a proper probe annotation for microarray platforms is an essential requirement to achieve good sensitivity in the identification of miRNA-mRNA interactions. We compare the results obtained from the analysis of the same expression profiles using both gene and transcript based custom CDFs that we have developed for a number of different annotations (ENSEMBL, RefSeq, AceView). In all cases, transcript-based annotations clearly improve the effectiveness of data integration and thus provide a more reliable confirmation of computationally predicted miRNA-mRNA interactions.
Biasiolo M, Forcato M, Possamai L, Ferrari F, Agnelli L, Lionetti M, Todoerti K, Neri A, Marchiori M, Bortoluzzi S, Bicciato S.
Critical analysis of transcriptional and post-transcriptional regulatory networks in multiple myeloma.
Pac Symp Biocomput. 2010:397-408. Abstract
Network analysis has emerged as a powerful approach to understand complex phenomena and organization in social, technological and biological systems. In particular, it is increasingly recognized the role played by the topology of cellular networks, the intricate web of interactions among genes, proteins and other molecules regulating cell activity, in unveiling the biological mechanisms underlying the physiological states of living organisms. In this study, critical analysis of network components has been applied to inspect the transcriptional and post-transcriptional regulatory networks reconstructed from mRNA and microRNA expression data of multiple myeloma (MM) samples. Specifically, the importance of a gene as a putative regulatory element has been assessed calculating the drop in the network performance caused by its deactivation instead of quantifying its degree of connectivity. The application of critical analysis to transcriptional and post-transcriptional regulatory networks allowed inferring novel regulatory relations potentially functional in multiple myeloma.
Lionetti M, Biasiolo M, Agnelli L, Todoerti K, Mosca L, Fabris S, Sales G, Deliliers GL, Bicciato S, Lombardi L, Bortoluzzi S, Neri A.
Identification of microRNA expression patterns and definition of a microRNA/mRNA regulatory network in distinct molecular groups of multiple myeloma.
Blood. 2009 Dec 10;114(25):e20-6. doi: 10.1182/blood-2009-08-237495. Abstract
To date, little evidence of miRNA expression/deregulation in multiple myeloma has been reported. To characterize miRNA in the context of the major multiple myeloma molecular types, we generated miRNA expression profiles of highly purified malignant plasma cells from 40 primary tumors. Furthermore, transcriptional profiles, available for all patients, were used to investigate the occurrence of miRNA/predicted target mRNA pair anticorrelations, and the miRNA and genome-wide DNA data were integrated in a subset of patients to evaluate the influence of allelic imbalances on miRNA expression. Differential miRNA expression patterns were identified, which were mainly associated with the major IGH translocations; particularly, t(4;14) patients showed specific overexpression of let-7e, miR-125a-5p, and miR-99b belonging to a cluster at 19q13.33. The occurrence of other lesions (ie, 1q gain, 13q and 17p deletions, and hyperdiploidy) was slightly characterized by specific miRNA signatures. Furthermore, the occurrence of several allelic imbalances or loss of heterozygosity was found significantly associated with the altered expression of miRNAs located in the involved regions, such as let-7b at 22q13.31 or miR-140-3p at 16q22. Finally, the integrative analysis based on computational target prediction and miRNA/mRNA profiling defined a network of putative functional miRNA-target regulatory relations supported by expression data.
Nie H, Neerincx PB, van der Poel J, Ferrari F, Bicciato S, Leunissen JA, Groenen MA.
Microarray data mining using Bioconductor packages.
BMC Proc. 2009 Jul 16;3 Suppl 4:S9. doi: 10.1186/1753-6561-3-S4-S9. Abstract
This paper describes the results of a Gene Ontology (GO) term enrichment analysis of chicken microarray data using the Bioconductor packages. By checking the enriched GO terms in three contrasts, MM8-PM8, MM8-MA8, and MM8-MM24, of the provided microarray data during this workshop, this analysis aimed to investigate the host reactions in chickens occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. The results of GO enrichment analysis using GO terms annotated to chicken genes and GO terms annotated to chicken-human orthologous genes were also compared. Furthermore, a locally adaptive statistical procedure (LAP) was performed to test differentially expressed chromosomal regions, rather than individual genes, in the chicken genome after Eimeria challenge.
GO enrichment analysis identified significant (raw p-value < 0.05) GO terms for all three contrasts included in the analysis. Some of the GO terms linked to, generally, primary immune responses or secondary immune responses indicating the GO enrichment analysis is a useful approach to analyze microarray data. The comparisons of GO enrichment results using chicken gene information and chicken-human orthologous gene information showed more refined GO terms related to immune responses when using chicken-human orthologous gene information, this suggests that using chicken-human orthologous gene information has higher power to detect significant GO terms with more refined functionality. Furthermore, three chromosome regions were identified to be significantly up-regulated in contrast MM8-PM8 (q-value < 0.01).
Overall, this paper describes a practical approach to analyze microarray data in farm animals where the genome information is still incomplete. For farm animals, such as chicken, with currently limited gene annotation, borrowing gene annotation information from orthologous genes in well-annotated species, such as human, will help improve the pathway analysis results substantially. Furthermore, LAP analysis approach is a relatively new and very useful way to be applied in microarray analysis.
Hedegaard J, Arce C, Bicciato S, Bonnet A, Buitenhuis B, Collado-Romero M, Conley LN, Sancristobal M, Ferrari F, Garrido JJ, Groenen MA, Hornshøj H, Hulsegge I, Jiang L, Jiménez-Marín A, Kommadath A, Lagarrigue S, Leunissen JA, Liaubet L, Neerincx PB, Nie H, van der Poel J, Prickett D, Ramirez-Boo M, Rebel JM, Robert-Granié C, Skarman A, Smits MA, Sørensen P, Tosser-Klopp G, Watson M.
Methods for interpreting lists of affected genes obtained in a DNA microarray experiment.
BMC Proc. 2009 Jul 16;3 Suppl 4:S5. Abstract
The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria.
Several conceptually different analytical approaches, using both commercial and public available software, were applied by the participating groups. The following tools were used: Ingenuity Pathway Analysis, MAPPFinder, LIMMA, GOstats, GOEAST, GOTM, Globaltest, TopGO, ArrayUnlock, Pathway Studio, GIST and AnnotationDbi. The main focus of the approaches was to utilise the relation between probes/genes and their gene ontology and pathways to interpret the affected probes/genes. The lack of a well-annotated chicken genome did though limit the possibilities to fully explore the tools. The main results from these analyses showed that the biological interpretation is highly dependent on the statistical method used but that some common biological conclusions could be reached.
It is highly recommended to test different analytical methods on the same data set and compare the results to obtain a reliable biological interpretation of the affected genes in a DNA microarray experiment.
Bisognin A, Coppe A, Ferrari F, Risso D, Romualdi C, Bicciato S, Bortoluzzi S.
A-MADMAN: annotation-based microarray data meta-analysis tool.
BMC Bioinformatics. 2009 Jun 29;10:201. doi: 10.1186/1471-2105-10-201. Abstract
Publicly available datasets of microarray gene expression signals represent an unprecedented opportunity for extracting genomic relevant information and validating biological hypotheses. However, the exploitation of this exceptionally rich mine of information is still hampered by the lack of appropriate computational tools, able to overcome the critical issues raised by meta-analysis.
This work presents A-MADMAN, an open source web application which allows the retrieval, annotation, organization and meta-analysis of gene expression datasets obtained from Gene Expression Omnibus. A-MADMAN addresses and resolves several open issues in the meta-analysis of gene expression data.
A-MADMAN allows i) the batch retrieval from Gene Expression Omnibus and the local organization of raw data files and of any related meta-information, ii) the re-annotation of samples to fix incomplete, or otherwise inadequate, metadata and to create user-defined batches of data, iii) the integrative analysis of data obtained from different Affymetrix platforms through custom chip definition files and meta-normalization. Software and documentation are available on-line at http://compgen.bio.unipd.it/bioinfo/amadman/.
Bicciato S, Spinelli R, Zampieri M, Mangano E, Ferrari F, Beltrame L, Cifola I, Peano C, Solari A, Battaglia C.
A computational procedure to identify significant overlap of differentially expressed and genomic imbalanced regions in cancer datasets.
Nucleic Acids Res. 2009 Aug;37(15):5057-70. doi: 10.1093/nar/gkp520. Epub 2009 Jun 19. Abstract
The integration of high-throughput genomic data represents an opportunity for deciphering the interplay between structural and functional organization of genomes and for discovering novel biomarkers. However, the development of integrative approaches to complement gene expression (GE) data with other types of gene information, such as copy number (CN) and chromosomal localization, still represents a computational challenge in the genomic arena. This work presents a computational procedure that directly integrates CN and GE profiles at genome-wide level. When applied to DNA/RNA paired data, this approach leads to the identification of Significant Overlaps of Differentially Expressed and Genomic Imbalanced Regions (SODEGIR). This goal is accomplished in three steps. The first step extends to CN a method for detecting regional imbalances in GE. The second part provides the integration of CN and GE data and identifies chromosomal regions with concordantly altered genomic and transcriptional status in a tumor sample. The last step elevates the single-sample analysis to an entire dataset of tumor specimens. When applied to study chromosomal aberrations in a collection of astrocytoma and renal carcinoma samples, the procedure proved to be effective in identifying discrete chromosomal regions of coordinated CN alterations and changes in transcriptional levels.
Adorno M, Cordenonsi M, Montagner M, Dupont S, Wong C, Hann B, Solari A, Bobisse S, Rondina MB, Guzzardo V, Parenti AR, Rosato A, Bicciato S, Balmain A, Piccolo S.
A Mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis.
Cell. 2009 Apr 3;137(1):87-98. doi: 10.1016/j.cell.2009.01.039. Abstract
TGFbeta ligands act as tumor suppressors in early stage tumors but are paradoxically diverted into potent prometastatic factors in advanced cancers. The molecular nature of this switch remains enigmatic. Here, we show that TGFbeta-dependent cell migration, invasion and metastasis are empowered by mutant-p53 and opposed by p63. Mechanistically, TGFbeta acts in concert with oncogenic Ras and mutant-p53 to induce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essential platforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63, we identified two candidate metastasis suppressor genes associated with metastasis risk in a large cohort of breast cancer patients. Thus, two common oncogenic lesions, mutant-p53 and Ras, selected in early neoplasms to promote growth and survival, also prefigure a cellular set-up with particular metastasis proclivity by TGFbeta-dependent inhibition of p63 function.
Rodilla V1, Villanueva A, Obrador-Hevia A, Robert-Moreno A, Fernández-Majada V, Grilli A, López-Bigas N, Bellora N, Albà MM, Torres F, Duñach M, Sanjuan X, Gonzalez S, Gridley T, Capella G, Bigas A, Espinosa L.
Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer.
Proc Natl Acad Sci U S A. 2009 Apr 14;106(15):6315-20. doi: 10.1073/pnas.0813221106. Epub 2009 Mar 26. Abstract
Notch has been linked to beta-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/beta-catenin (down-regulated when blocking Wnt/beta-catenin) that are directly regulated by Notch (repressed by gamma-secretase inhibitors and up-regulated by active Notch1 in the absence of beta-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through beta-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/beta-catenin pathway in tumors implanted s.c. in nude mice. Crossing APC(Min/+) with Jagged1(+/Delta) mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear beta-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by beta-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.
Carata E, Peano C, Tredici SM, Ferrari F, Talà A, Corti G, Bicciato S, De Bellis G, Alifano P.
Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production.
Microb Cell Fact. 2009 Mar 30;8:18. doi: 10.1186/1475-2859-8-18. Abstract
There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.
Spontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.
Rifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.
Coppe A, Ferrari F, Bisognin A, Danieli GA, Ferrari S, Bicciato S, Bortoluzzi S.
Motif discovery in promoters of genes co-localized and co-expressed during myeloid cells differentiation.
Nucleic Acids Res. 2009 Feb;37(2):533-49. doi: 10.1093/nar/gkn948. Epub 2008 Dec 5. Abstract
Genes co-expressed may be under similar promoter-based and/or position-based regulation. Although data on expression, position and function of human genes are available, their true integration still represents a challenge for computational biology, hampering the identification of regulatory mechanisms. We carried out an integrative analysis of genomic position, functional annotation and promoters of genes expressed in myeloid cells. Promoter analysis was conducted by a novel multi-step method for discovering putative regulatory elements, i.e. over-represented motifs, in a selected set of promoters, as compared with a background model. The combination of transcriptional, structural and functional data allowed the identification of sets of promoters pertaining to groups of genes co-expressed and co-localized in regions of the human genome. The application of motif discovery to 26 groups of genes co-expressed in myeloid cells differentiation and co-localized in the genome showed that there are more over-represented motifs in promoters of co-expressed and co-localized genes than in promoters of simply co-expressed genes (CEG). Motifs, which are similar to the binding sequences of known transcription factors, non-uniformly distributed along promoter sequences and/or occurring in highly co-expressed subset of genes were identified. Co-expressed and co-localized gene sets were grouped in two co-expressed genomic meta-regions, putatively representing functional domains of a high-level expression regulation.
Bungaro S, Dell’Orto MC, Zangrando A, Basso D, Gorletta T, Lo Nigro L, Leszl A, Young BD, Basso G, Bicciato S, Biondi A, te Kronnie G, Cazzaniga G.
Integration of genomic and gene expression data of childhood ALL without known aberrations identifies subgroups with specific genetic hallmarks.
Genes Chromosomes Cancer. 2009 Jan;48(1):22-38. doi: 10.1002/gcc.20616. Abstract
Pediatric acute lymphoblastic leukemia (ALL) comprises genetically distinct subtypes. However, 25% of cases still lack defined genetic hallmarks. To identify genomic aberrancies in childhood ALL patients nonclassifiable by conventional methods, we performed a single nucleotide polymorphisms (SNP) array-based genomic analysis of leukemic cells from 29 cases. The vast majority of cases analyzed (19/24, 79%) showed genomic abnormalities; at least one of them affected either genes involved in cell cycle regulation or in B-cell development. The most relevant abnormalities were CDKN2A/9p21 deletions (7/24, 29%), ETV6 (TEL)/12p13 deletions (3/24, 12%), and intrachromosomal amplifications of chromosome 21 (iAMP21) (3/24, 12%). To identify variation in expression of genes directly or indirectly affected by recurrent genomic alterations, we integrated genomic and gene expression data generated by microarray analyses of the same samples. SMAD1 emerged as a down-regulated gene in CDKN2A homozygous deleted cases compared with nondeleted. The JAG1 gene, encoding the Jagged 1 ligand of the Notch receptor, was among a list of differentially expressed (up-regulated) genes in ETV6-deleted cases. Our findings demonstrate that integration of genomic analysis and gene expression profiling can identify genetic lesions undetected by routine methods and potential novel pathways involved in B-progenitor ALL pathogenesis.
Rizzi N, Liu N, Napolitano C, Nori A, Turcato F, Colombi B, Bicciato S, Arcelli D, Spedito A, Scelsi M, Villani L, Esposito G, Boncompagni S, Protasi F, Volpe P, Priori SG.
Unexpected structural and functional consequences of the R33Q homozygous mutation in cardiac calsequestrin: a complex arrhythmogenic cascade in a knock in mouse model.
Circ Res. 2008 Aug 1;103(3):298-306. doi: 10.1161/CIRCRESAHA.108.171660. Epub 2008 Jun 26. Abstract
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disorder characterized by life threatening arrhythmias elicited by physical and emotional stress in young individuals. The recessive form of CPVT is associated with mutation in the cardiac calsequestrin gene (CASQ2). We engineered and characterized a homozygous CASQ2(R33Q/R33Q) mouse model that closely mimics the clinical phenotype of CPVT patients. CASQ2(R33Q/R33Q) mice develop bidirectional VT on exposure to environmental stress whereas CASQ2(R33Q/R33Q) myocytes show reduction of the sarcoplasmic reticulum (SR) calcium content, adrenergically mediated delayed (DADs) and early (EADs) afterdepolarizations leading to triggered activity. Furthermore triadin, junctin, and CASQ2-R33Q proteins are significantly decreased in knock-in mice despite normal levels of mRNA, whereas the ryanodine receptor (RyR2), calreticulin, phospholamban, and SERCA2a-ATPase are not changed. Trypsin digestion studies show increased susceptibility to proteolysis of mutant CASQ2. Despite normal histology, CASQ2(R33Q/R33Q) hearts display ultrastructural changes such as disarray of junctional electron-dense material, referable to CASQ2 polymers, dilatation of junctional SR, yet normal total SR volume. Based on the foregoings, we propose that the phenotype of the CASQ2(R33Q/R33Q) CPVT mouse model is portrayed by an unexpected set of abnormalities including (1) reduced CASQ2 content, possibly attributable to increased degradation of CASQ2-R33Q, (2) reduction of SR calcium content, (3) dilatation of junctional SR, and (4) impaired clustering of mutant CASQ2.
Peano C, Bicciato S, Corti G, Ferrari F, Rizzi E, Bonnal RJ, Bordoni R, Albertini A, Bernardi LR, Donadio S, De Bellis G.
Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays.
Microb Cell Fact. 2007 Nov 26;6:37. Abstract
The Saccharopolyspora erythraea genome sequence, recently published, presents considerable divergence from those of streptomycetes in gene organization and function, confirming the remarkable potential of S. erythraea for producing many other secondary metabolites in addition to erythromycin. In order to investigate, at whole transcriptome level, how S. erythraea genes are modulated, a DNA microarray was specifically designed and constructed on the S. erythraea strain NRRL 2338 genome sequence, and the expression profiles of 6494 ORFs were monitored during growth in complex liquid medium.
The transcriptional analysis identified a set of 404 genes, whose transcriptional signals vary during growth and characterize three distinct phases: a rapid growth until 32 h (Phase A); a growth slowdown until 52 h (Phase B); and another rapid growth phase from 56 h to 72 h (Phase C) before the cells enter the stationary phase. A non-parametric statistical method, that identifies chromosomal regions with transcriptional imbalances, determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, and strand specific patterns of expression. Microarray data were used to characterize the temporal behaviour of major functional classes and of all the gene clusters for secondary metabolism. The results confirmed that the ery cluster is up-regulated during Phase A and identified six additional clusters (for terpenes and non-ribosomal peptides) that are clearly regulated in later phases.
The use of a S. erythraea DNA microarray improved specificity and sensitivity of gene expression analysis, allowing a global and at the same time detailed picture of how S. erythraea genes are modulated. This work underlines the importance of using DNA microarrays, coupled with an exhaustive statistical and bioinformatic analysis of the results, to understand the transcriptional organization of the chromosomes of micro-organisms producing natural products.
Ferrari F, Bortoluzzi S, Coppe A, Sirota A, Safran M, Shmoish M, Ferrari S, Lancet D, Danieli GA, Bicciato S.
Novel definition files for human GeneChips based on GeneAnnot.
BMC Bioinformatics. 2007 Nov 15;8:446. Abstract
Improvements in genome sequence annotation revealed discrepancies in the original probeset/gene assignment in Affymetrix microarray and the existence of differences between annotations and effective alignments of probes and transcription products. In the current generation of Affymetrix human GeneChips, most probesets include probes matching transcripts from more than one gene and probes which do not match any transcribed sequence.
We developed a novel set of custom Chip Definition Files (CDF) and the corresponding Bioconductor libraries for Affymetrix human GeneChips, based on the information contained in the GeneAnnot database. GeneAnnot-based CDFs are composed of unique custom-probesets, including only probes matching a single gene.
GeneAnnot-based custom CDFs solve the problem of a reliable reconstruction of expression levels and eliminate the existence of more than one probeset per gene, which often leads to discordant expression signals for the same transcript when gene differential expression is the focus of the analysis. GeneAnnot CDFs are freely distributed and fully compliant with Affymetrix standards and all available software for gene expression analysis. The CDF libraries are available from http://www.xlab.unimo.it/GA_CDF, along with supplementary information (CDF libraries, installation guidelines and R code, CDF statistics, and analysis results).
Ferrari F, Bortoluzzi S, Coppe A, Basso D, Bicciato S, Zini R, Gemelli C, Danieli GA, Ferrari S.
Genomic expression during human myelopoiesis.
BMC Genomics. 2007 Aug 3;8:264. Abstract
Human myelopoiesis is an exciting biological model for cellular differentiation since it represents a plastic process where multipotent stem cells gradually limit their differentiation potential, generating different precursor cells which finally evolve into distinct terminally differentiated cells. This study aimed at investigating the genomic expression during myeloid differentiation through a computational approach that integrates gene expression profiles with functional information and genome organization.
Gene expression data from 24 experiments for 8 different cell types of the human myelopoietic lineage were used to generate an integrated myelopoiesis dataset of 9,425 genes, each reliably associated to a unique genomic position and chromosomal coordinate. Lists of genes constitutively expressed or silent during myelopoiesis and of genes differentially expressed in commitment phase of myelopoiesis were first identified using a classical data analysis procedure. Then, the genomic distribution of myelopoiesis genes was investigated integrating transcriptional and functional characteristics of genes. This approach allowed identifying specific chromosomal regions significantly highly or weakly expressed, and clusters of differentially expressed genes and of transcripts related to specific functional modules.
The analysis of genomic expression during human myelopoiesis using an integrative computational approach allowed discovering important relationships between genomic position, biological function and expression patterns and highlighting chromatin domains, including genes with coordinated expression and lineage-specific functions.
Agnelli L, Fabris S, Bicciato S, Basso D, Baldini L, Morabito F, Verdelli D, Todoerti K, Lambertenghi-Deliliers G, Lombardi L, Neri A.
Upregulation of translational machinery and distinct genetic subgroups characterise hyperdiploidy in multiple myeloma.
Br J Haematol. 2007 Feb;136(4):565-73. Abstract
Karyotypic instability, including numerical and structural chromosomal aberrations, represents a distinct feature of multiple myeloma (MM). About 40-50% of patients display hyperdiploidy, defined by recurrent trisomies of non-random chromosomes. To molecularly characterise hyperdiploid (H) and nonhyperdiploid (NH) MM, we analysed the gene expression profiles of 66 primary tumours, and used fluorescence in situ hybridisation to investigate the major chromosomal alterations. The differential expression of 225 genes mainly involved in protein biosynthesis, transcriptional machinery and oxidative phosphorylation distinguished the 28 H-MM from the 38 NH-MM cases. The 204 upregulated genes in H-MM mapped mainly to the chromosomes involved in hyperdiploidy, and the 29% upregulated genes in NH-MM mapped to 16q. The identified transcriptional fingerprint was robustly validated on a publicly available gene expression dataset of 64 MM cases; and the global expression modulation of regions on the chromosomes involved in hyperdiploidy was verified using a self-developed non-parametric statistical method. H-MM could be further divided into two distinct molecular and transcriptional entities, characterised by the presence of trisomy 11 and 1q-extracopies/chromosome 13 deletion respectively. These data reinforce the importance of combining molecular cytogenetics and gene expression profiling to define a genomic framework for the study of MM pathogenesis and clinical management.
Agnelli L, Bicciato S, Fabris S, Baldini L, Morabito F, Intini D, Verdelli D, Callegaro A, Bertoni F, Lambertenghi-Deliliers G, Lombardi L, Neri A.
Integrative genomic analysis reveals distinct transcriptional and genetic features associated with chromosome 13 deletion in multiple myeloma.
Haematologica. 2007 Jan;92(1):56-65. Abstract
BACKGROUND AND OBJECTIVES:
The chromosome 13 deletion (Delta13) is one of the most frequent chromosomal alterations in multiple myeloma (MM). Delta13 is associated with an unfavorable prognosis, although there is increasing agreement that its prognostic relevance must be related to the ploidy status and the presence of different chromosomal translocations. The aim of this study was to provide a comprehensive analysis of the transcriptional features of Delta13 in MM.
DESIGN AND METHODS:
Highly purified plasma cells from 80 newly diagnosed MM patients were characterized by means of fluorescence in situ hybridization (FISH) and high-density oligonucleotide microarray for gene expression profiling and chromosomal alterations.
We identified 67 differentially expressed genes in the patients with and without the chromosome 13 deletion, all of which were downregulated in the cases with Delta13: 44 mapped along the whole chromosome 13, seven on chromosome 11 and three on chromosome 19. Functional analyses of the selected genes indicated their involvement in protein biosynthesis, ubiquitination and transcriptional regulation. An integrative genomic approach based on regional analyses of the gene expression data identified distinct chromosomal regions whose global expression modulation could differentiate Delta13-positive cases, in particular the upregulation of 1q21-1q42 and the downregulation of 19p and almost the entire chromosome 11. FISH analyses confirmed the close relationship between Delta13-positivity and the presence of extra copies of 1q21-1q42 (p=6 x 10(-4)) or the absence of chromosome 11 and 19 trisomy (p=5 x 10(-4)).
INTERPRETATION AND CONCLUSIONS:
Our results indicate that distinct types of chromosomal aberrations are closely related to the transcriptional profiles of Delta13-positive cases, suggesting that the contribution of Delta13 to the malignancy should be considered together with associated abnormalities.
Lombardi L, Poretti G, Mattioli M, Fabris S, Agnelli L, Bicciato S, Kwee I, Rinaldi A, Ronchetti D, Verdelli D, Lambertenghi-Deliliers G, Bertoni F, Neri A.
Molecular characterization of human multiple myeloma cell lines by integrative genomics: insights into the biology of the disease.
Genes Chromosomes Cancer. 2007 Mar;46(3):226-38. Abstract
To investigate the patterns of genetic lesions in a panel of 23 human multiple myeloma cell lines (HMCLs), we made a genomic integrative analysis involving FISH, and both gene expression and genome-wide profiling approaches. The expression profiles of the genes targeted by the main IGH translocations showed that the WHSC1/MMSET gene involved in t(4;14)(p16;q32) was expressed at different levels in all of the HMCLs, and that the expression of the MAF gene was not restricted to the HMCLs carrying t(14;16)(q32;q23). Supervised analyses identified a limited number of genes specifically associated with t(4;14) and involved in different biological processes. The signature related to MAF/MAFB expression included the known MAF target genes CCND2 and ITGB7, as well as genes controlling cell shape and cell adhesion. Genome-wide DNA profiling allowed the identification of a gain on chromosome arm 1q in 88% of the analyzed cell lines, together with recurrent gains on 8q, 18q, 7q, and 20q; the most frequent deletions affected 1p, 13q, 17p, and 14q; and almost all of the cell lines presented LOH on chromosome 13. Two hundred and twenty-two genes were found to be simultaneously overexpressed and amplified in our panel, including the BCL2 locus at 18q21.33. Our data further support the evidence of the genomic complexity of multiple myeloma and reinforce the role of an integrated genomic approach in improving our understanding of the molecular pathogenesis of the disease. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.
Mandruzzato S, Callegaro A, Turcatel G, Francescato S, Montesco MC, Chiarion-Sileni V, Mocellin S, Rossi CR, Bicciato S, Wang E, Marincola FM, Zanovello P.
A gene expression signature associated with survival in metastatic melanoma.
J Transl Med. 2006 Nov 27;4:50. Abstract
Current clinical and histopathological criteria used to define the prognosis of melanoma patients are inadequate for accurate prediction of clinical outcome. We investigated whether genome screening by means of high-throughput gene microarray might provide clinically useful information on patient survival.
Forty-three tumor tissues from 38 patients with stage III and stage IV melanoma were profiled with a 17,500 element cDNA microarray. Expression data were analyzed using significance analysis of microarrays (SAM) to identify genes associated with patient survival, and supervised principal components (SPC) to determine survival prediction.
SAM analysis revealed a set of 80 probes, corresponding to 70 genes, associated with survival, i.e. 45 probes characterizing longer and 35 shorter survival times, respectively. These transcripts were included in a survival prediction model designed using SPC and cross-validation which allowed identifying 30 predicting probes out of the 80 associated with survival.
The longer-survival group of genes included those expressed in immune cells, both innate and acquired, confirming the interplay between immunological mechanisms and the natural history of melanoma. Genes linked to immune cells were totally lacking in the poor-survival group, which was instead associated with a number of genes related to highly proliferative and invasive tumor cells.
Gallina G, Dolcetti L, Serafini P, De Santo C, Marigo I, Colombo MP, Basso G, Brombacher F, Borrello I, Zanovello P, Bicciato S, Bronte V.
Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells.
J Clin Invest. 2006 Oct;116(10):2777-90. Abstract
Active suppression of tumor-specific T lymphocytes can limit the efficacy of immune surveillance and immunotherapy. While tumor-recruited CD11b+ myeloid cells are known mediators of tumor-associated immune dysfunction, the true nature of these suppressive cells and the fine biochemical pathways governing their immunosuppressive activity remain elusive. Here we describe a population of circulating CD11b+IL-4 receptor alpha+ (CD11b+IL-4Ralpha+), inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-gamma released from T lymphocytes. CD11b+IL-4Ralpha+ cells produced IL-13 and IFN-gamma and integrated the downstream signals of these cytokines to trigger the molecular pathways suppressing antigen-activated CD8+ T lymphocytes. Analogous immunosuppressive circuits were active in CD11b+ cells present within the tumor microenvironment. These suppressor cells challenge the current idea that tumor-conditioned immunosuppressive monocytes/macrophages are alternatively activated. Moreover, our data show how the inflammatory response elicited by tumors had detrimental effects on the adaptive immune system and suggest novel approaches for the treatment of tumor-induced immune dysfunctions.
Severgnini M, Bicciato S, Mangano E, Scarlatti F, Mezzelani A, Mattioli M, Ghidoni R, Peano C, Bonnal R, Viti F, Milanesi L, De Bellis G, Battaglia C.
Strategies for comparing gene expression profiles from different microarray platforms: application to a case-control experiment.
Anal Biochem. 2006 Jun 1;353(1):43-56. Epub 2006 Apr 3. Abstract
Meta-analysis of microarray data is increasingly important, considering both the availability of multiple platforms using disparate technologies and the accumulation in public repositories of data sets from different laboratories. We addressed the issue of comparing gene expression profiles from two microarray platforms by devising a standardized investigative strategy. We tested this procedure by studying MDA-MB-231 cells, which undergo apoptosis on treatment with resveratrol. Gene expression profiles were obtained using high-density, short-oligonucleotide, single-color microarray platforms: GeneChip (Affymetrix) and CodeLink (Amersham). Interplatform analyses were carried out on 8414 common transcripts represented on both platforms, as identified by LocusLink ID, representing 70.8% and 88.6% of annotated GeneChip and CodeLink features, respectively. We identified 105 differentially expressed genes (DEGs) on CodeLink and 42 DEGs on GeneChip. Among them, only 9 DEGs were commonly identified by both platforms. Multiple analyses (BLAST alignment of probes with target sequences, gene ontology, literature mining, and quantitative real-time PCR) permitted us to investigate the factors contributing to the generation of platform-dependent results in single-color microarray experiments. An effective approach to cross-platform comparison involves microarrays of similar technologies, samples prepared by identical methods, and a standardized battery of bioinformatic and statistical analyses.
Te Kronnie G, Bicciato S, Franceschini L, Accordi B, Dellíorto MC, Rinaldi A, Pession A, Barisone E, Conter V, Locatelli F, Basso G.
Validation by RQ-PCR and flow cytometry of alpha-defensin1-3 (DEFA1-3) overexpression in relapsed and refractory acute lymphoblastic leukemia.
Oncol Rep. 2006 Feb;15(2):341-6. Abstract
In spite of high cure rates and improved overall survival, 25% of pediatric patients with acute lymphoblastic leukemia (ALL) relapse after obtaining complete remission. Additionally a small proportion of patients are refractory and do not attain remission. Microarray expression analysis of matched diagnosis-relapse B-lineage ALL sample pairs identified DEFA1-3 as a potential marker of relapse. Here, validation of DEFA1-3 as a marker for therapy resistance is explored. DEFA1-3 expression was analysed by RQ-PCR in patient paired samples at diagnosis and relapse of 6 early-relapse (within 18 months) and 8 late-relapse (beyond 18 months) B-lineage ALL. Diagnostic samples of 19 patients with ALL who are in continuous complete remission (median time from diagnosis 47 month) and diagnostic samples of 5 refractory patients who had not achieved remission at day 35 of therapy were also analyzed. In addition, overexpression of alpha-defensin1-3 proteins in blast cells at relapse was analysed by flow cytometry. DEFA1-3 was overexpressed at relapse as compared to diagnosis in 12 of 14 samples. At diagnosis, the expression of DEFA1-3 was significantly higher in samples from refractory patients as compared to those of patients who are in CR and to those of patients who experienced late relapse. At diagnosis, patients who relapsed early after diagnosis could not be distinguished from refractory patients based on DEFA1-3 expression levels. Results suggest that high levels of DEFA1-3 mRNA and alpha-defensin1-3 protein expression are correlated with disease progression and failure of adequate response to conventional chemotherapy.
Orabona C, Puccetti P, Vacca C, Bicciato S, Luchini A, Fallarino F, Bianchi R, Velardi E, Perruccio K, Velardi A, Bronte V, Fioretti MC, Grohmann U.
Toward the identification of a tolerogenic signature in IDO-competent dendritic cells.
Blood. 2006 Apr 1;107(7):2846-54. Epub 2005 Dec 8. Erratum in: Blood. 2006 May 1;107(9):3441. Abstract
Although much is known about the transcriptional profiles of dendritic cells (DCs) during maturation, the molecular switches critical for the induction of a tolerogenic program in DC subsets are still obscure. We examined the gene-expression profiles of murine splenic CD8+ DCs rendered highly tolerogenic by interferon-gamma (IFN-gamma), which activates the enzyme indoleamine 2,3-dioxygenase (IDO, encoded by Indo) and thus initiates the immunosuppressive pathway of tryptophan catabolism. By examining the expression of a series of relevant genes in IDO+ compared with IDO- DCs, we found consistent and selective association of the IDO-competent phenotype with down-modulation of the Tyrobp gene, encoding the signaling adapter DAP12, which typically associates with activating receptors. Down-modulation of Tyrobp involved IFN consensus sequence binding protein (ICSBP), a transcription factor also known as IRF-8. In murine and human monocyte-derived DCs, silencing DAP12 expression imparted IDO functional competence to IDO- cells, whereas silencing IRF-8 in IDO+ counterparts abolished IDO expression and function. Thus, IRF-8 is required in tolerogenic DCs for the positive regulation of Indo and the negative regulation of Tyrobp. Overall, these studies reveal the occurrence of a simple and evolutionarily conserved code in the control of tolerance by an ancestral metabolic enzyme.
Agnelli L, Bicciato S, Mattioli M, Fabris S, Intini D, Verdelli D, Baldini L, Morabito F, Callea V, Lombardi L, Neri A.
Molecular classification of multiple myeloma: a distinct transcriptional profile characterizes patients expressing CCND1 and negative for 14q32 translocations.
J Clin Oncol. 2005 Oct 10;23(29):7296-306. Epub 2005 Aug 29. Abstract
The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM). A recently proposed classification grouped MM patients into five classes on the basis of their cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32. In this study, we provide a molecular characterization of the identified translocations/cyclins (TC) groups.
MATERIALS AND METHODS:
The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes and identify their transcriptional fingerprints. The cyclin D expression data were validated by means of real-time quantitative polymerase chain reaction analysis; fluorescence in situ hybridization was used to investigate the cyclin D loci arrangements, and to detect the main IGH translocations and the chromosome 13q deletion.
Class-prediction analysis identified 112 probe sets as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. The TC2 group, which showed extra copies of the CCND1 locus and no IGH translocations or the chromosome 13q deletion, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis of published data sets validated the identified gene expression signatures.
Our data contribute to the understanding of the molecular and biologic features of distinct MM subtypes. The identification of a distinctive gene expression pattern in TC2 patients may improve risk stratification and indicate novel therapeutic targets.
Vacca A, Scavelli C, Montefusco V, Di Pietro G, Neri A, Mattioli M, Bicciato S, Nico B, Ribatti D, Dammacco F, Corradini P.
Thalidomide downregulates angiogenic genes in bone marrow endothelial cells of patients with active multiple myeloma.
J Clin Oncol. 2005 Aug 10;23(23):5334-46. Epub 2005 Jun 6. Abstract
To study the antiangiogenic effect of thalidomide.
PATIENTS AND METHODS:
The expression of key angiogenic genes was studied in bone marrow endothelial cells (ECs) of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies unattributed/unassociated (MG[u]), diffuse large B-cell non-Hodgkin’s lymphoma, in a Kaposi’s sarcoma (KS) cell line, and in healthy human umbilical vein ECs (HUVECs) following exposure to therapeutic doses of thalidomide.
Thalidomide markedly downregulates the genes in a dose-dependent fashion in active MMECs and KS cell line, but upregulates them or is ineffective in nonactive MMECs, MG(u)ECs, NHL-ECs, and in HUVECs. Secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor also diminishes according to the dose in culture conditioned media (CM) of active MMECs and KS, whereas it does not change in the other CM.
Inhibition by thalidomide is probably confined to the genes of active MMECs and KS. This would account for its higher efficacy in these diseases.
Mattioli M, Agnelli L, Fabris S, Baldini L, Morabito F, Bicciato S, Verdelli D, Intini D, Nobili L, Cro L, Pruneri G, Callea V, Stelitano C, Maiolo AT, Lombardi L, Neri A.
Gene expression profiling of plasma cell dyscrasias reveals molecular patterns associated with distinct IGH translocations in multiple myeloma.
Oncogene. 2005 Apr 7;24(15):2461-73. Abstract
Multiple myeloma (MM) is the most common form of plasma cell dyscrasia, characterized by a marked heterogeneity of genetic lesions and clinical course. It may develop from a premalignant condition (monoclonal gammopathy of undetermined significance, MGUS) or progress from intramedullary to extramedullary forms (plasma cell leukemia, PCL). To provide insights into the molecular characterization of plasma cell dyscrasias and to investigate the contribution of specific genetic lesions to the biological and clinical heterogeneity of MM, we analysed the gene expression profiles of plasma cells isolated from seven MGUS, 39 MM and six PCL patients by means of DNA microarrays. MMs resulted highly heterogeneous at transcriptional level, whereas the differential expression of genes mainly involved in DNA metabolism and proliferation distinguished MGUS from PCLs and the majority of MM cases. The clustering of MM patients was mainly driven by the presence of the most recurrent translocations involving the immunoglobulin heavy-chain locus. Distinct gene expression patterns have been found to be associated with different lesions: the overexpression of CCND2 and genes involved in cell adhesion pathways was observed in cases with deregulated MAF and MAFB, whereas genes upregulated in cases with the t(4;14) showed apoptosis-related functions. The peculiar finding in patients with the t(11;14) was the downregulation of the alpha-subunit of the IL-6 receptor. In addition, we identified a set of cancer germline antigens specifically expressed in a subgroup of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.
Artificial neural network technologies to identify biomarkers for therapeutic intervention.
Curr Opin Mol Ther. 2004 Dec;6(6):616-23. Review. Abstract
High-throughput technologies such as DNA/RNA microarrays, mass spectrometry and protein chips are creating unprecedented opportunities to accelerate towards the understanding of living systems and the identification of target genes and pathways for drug development and therapeutic intervention. However, the increasing volumes of data generated by molecular profiling experiments pose formidable challenges to investigate an overwhelming mass of information and turn it into predictive, deployable markers. Advanced biostatistics and machine learning methods from computer science have been applied to analyze and correlate numerical values of profiling intensities to physiological states. This article reviews the application of artificial neural networks, an information-processing tool, to the identification of sets of diagnostic/prognostic biomarkers from high-throughput profiling data.
Te Kronnie G, Bicciato S, Basso G.
Acute leukemia subclassification: a marker protein expression perspective.
Hematology. 2004 Jun;9(3):165-70. Review. Abstract
Improved leukemia classification and tailoring of therapy have greatly improved patient outcome particularly for children with acute leukemia (AL). Using immunophenotyping, molecular genetics and cytogenetics the low hanging fruits of biomedical research have been successfully incorporated in routine diagnosis of leukemia subclasses. Future improvements in the classification and understanding of leukemia biology will very likely be more slow and laborious. Recently, gene expression profiling has provided a framework for the global molecular analysis of hematological cancers, and high throughput proteomic analysis of leukemia samples is on the way. Here we consider classification of acute leukemia samples by flow cytometry using the marker proteins of immunophenotyping as a component of the proteome. Marker protein expressions are converted into quantitative expression values and subjected to computational analysis. Quantitative multivariate analysis from panels of marker proteins has demonstrated that marker protein expression profiles can distinguish MLLre from non-MLLre ALL cases and also allow to specifically distinguish MLL/AF4 cases. Potentially, these quantitative expression analyses can be used in clinical diagnosis. Immunophenotypic data collection using flow cytometry is a fast and relatively easily accessible technology that has already been implemented in most centers for leukemia diagnosis and the translation into quantitative expression data sets is a matter of flow cytometer settings and output calibration. However, before application in clinical diagnostics can occur it is crucial that quantitative immunophenotypic data set analysis is validated in independent experiments and in large data sets.
Dettin M, Bicciato S, Scarinci C, Cline E, Lingen MW, Di Bello C.
Synthetic peptides derived from the angiostatin K4 domain inhibit endothelial cell migration.
Chembiochem. 2003 Nov 7;4(11):1238-42. Abstract
Loop the loop: Several linear and cyclic peptides patterned on the angiostatin K4 domain (see picture) have been synthesized and tested in order to identify active sequence candidates for further studies aimed at elucidating the antimigratory activity and functionality of the K4 domain. Migration assays indicate that different K4 regions induce different degrees of inhibition of cell motility. In particular, the linear peptide reproducing the (358–379) fragment of angiostatin exhibits an inhibitory activity comparable to that of the entire K4 domain.
Hasina R, Hulett K, Bicciato S, Di Bello C, Petruzzelli GJ, Lingen MW.
Plasminogen activator inhibitor-2: a molecular biomarker for head and neck cancer progression.
Cancer Res. 2003 Feb 1;63(3):555-9. Abstract
Head and neck squamous cell carcinoma (HNSCC) is an aggressive epithelial malignancy in which the early diagnosis of premalignant lesions is known to directly correlate with increased survival. However, only a portion of biopsies showing dysplasia will progress to cancer, and there are no currently accepted criteria for predicting which lesions will progress. Therefore, diagnostic protocols that can identify the lesions that are likely to become HNSCC are required. RNA was isolated from normal keratinocytes, the immortalized but nontumorigenic HaCat cell line, and the tumor cell lines SCC-4, SCC-9, SCC-25, and OSCC-3. The RNA was then labeled and used to probe nylon microarray filters that contained a total of 9184 genes (5295 named and 3889 Expression Sequence Tags). Genes whose expression demonstrated a 3-fold or greater change were considered significant. Comparison of expression profiles from normal, HaCat, and four tumor cell lines revealed changes in gene expression in a total of 508 genes. Of these, 16 genes showed a consistent loss of expression when comparing normal to immortalized keratinocytes. In addition, 10 genes demonstrated a consistent loss of expression in the tumor cell lines only. In this latter group of genes, plasminogen activator inhibitor-2 (PAI-2), a gene whose expression has been linked to cell invasion, was additionally investigated. Altered expression of PAI-2 in the different cultured cells was validated via real-time quantitative-PCR. In addition, immunohistochemical evaluation of biopsy samples revealed a high expression of PAI-2 in both normal and dysplastic epithelium with a marked decrease of expression in areas of the biopsies containing HNSCC. These data demonstrate that genomic profiling can then be used to identify potential genotypic/phenotypic biomarkers that may predict which dysplastic lesions are most likely to progress to HNSCC.
Bicciato S, Pandin M, Didonè G, Di Bello C.
Pattern identification and classification in gene expression data using an autoassociative neural network model.
Biotechnol Bioeng. 2003 Mar 5;81(5):594-606. Abstract
The application of DNA microarray technology for analysis of gene expression creates enormous opportunities to accelerate the pace in understanding living systems and identification of target genes and pathways for drug development and therapeutic intervention. Parallel monitoring of the expression profiles of thousands of genes seems particularly promising for a deeper understanding of cancer biology and the identification of molecular signatures supporting the histological classification schemes of neoplastic specimens. However, the increasing volume of data generated by microarray experiments poses the challenge of developing equally efficient methods and analysis procedures to extract, interpret, and upgrade the information content of these databases. Herein, a computational procedure for pattern identification, feature extraction, and classification of gene expression data through the analysis of an autoassociative neural network model is described. The identified patterns and features contain critical information about gene-phenotype relationships observed during changes in cell physiology. They represent a rational and dimensionally reduced base for understanding the basic biology of the onset of diseases, defining targets of therapeutic intervention, and developing diagnostic tools for the identification and classification of pathological states. The proposed method has been tested on two different microarray datasets-Golub’s analysis of acute human leukemia [Golub et al. (1999) Science 286:531-537], and the human colon adenocarcinoma study presented by Alon et al. [1999; Proc Natl Acad Sci USA 97:10101-10106]. The analysis of the neural network internal structure allows the identification of specific phenotype markers and the extraction of peculiar associations among genes and physiological states. At the same time, the neural network outputs provide assignment to multiple classes, such as different pathological conditions or tissue samples, for previously unseen instances.
Cline EI, Bicciato S, DiBello C, Lingen MW.
Prediction of in vivo synergistic activity of antiangiogenic compounds by gene expression profiling.
Cancer Res. 2002 Dec 15;62(24):7143-8. Abstract
Angiogenesis, an essential phenotype for tumor formation, requires the interaction of many cells within the tumor microenvironment. Therefore, successful antiangiogenic therapies must be able to block all of the different mechanisms tumors use to induce neovascularization. A major challenge for developing such protocols is determining which agents are likely to have the highest degree of synergistic activity in vivo. We treated human microvascular endothelial cells with six inhibitors of angiogenesis and used microarrays to seek divergent patterns of gene expression suggestive of potential synergies. The expression profiles of a thrombospondin-mimetic peptide (DI-TSPa) and TNP-470 (TNP) were very similar, whereas endostatin had a dramatically different profile. In vitro, endostatin was synergistically antiangiogenic with either TNP-470 or DI-TSPa. In vivo, mice bearing Lewis lung carcinoma cells treated with a combination of endostatin and either DI-TSPa or TNP-470, at doses that were ineffective when used alone, resulted in a marked inhibition of tumor growth and decreased tumor angiogenesis. Conversely, animals treated with both DI-TSPa and TNP-470 demonstrated a modest effect on both tumor growth and angiogenesis. These results suggest that even in the absence of a complete mechanistic understanding of how these inhibitors work, gene expression profiling may be used to predict synergistic antiangiogenic activity and thus maximize their antitumor efficacy.
Bicciato S, Bagno A, Soldà M, Manfredini R, Di Bello C.
Fermentation diagnosis by multivariate statistical analysis.
Appl Biochem Biotechnol. 2002 Jul-Dec;102-103(1-6):49-62. Abstract
During the course of fermentation, online measuring procedures able to estimate the performance of the current operation are highly desired. Unfortunately, the poor mechanistic understanding of most biologic systems hampers attempts at direct online evaluation of the bioprocess, which is further complicated by the lack of appropriate online sensors and the long lag time associated with offline assays. Quite often available data lack sufficient detail to be directly used, and after a cursory evaluation are stored away. However, these historic databases of process measurements may still retain some useful information. A multivariate statistical procedure has been applied for analyzing the measurement profiles acquired during the monitoring of several fed-batch fermentations for the production of erythromycin. Multivariate principal component analysis has been used to extract information from the multivariate historic database by projecting the process variables onto a low-dimensional space defined by the principal components. Thus, each fermentation is identified by a temporal profile in the principal component plane. The projections represent monitoring charts, consistent with the concept of statistical process control, which are useful for tracking the progress of each fermentation batch and identifying anomalous behaviors (process diagnosis and fault detection).