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Professore Ordinario presso: Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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- Apparecchiatura e processo per la sintesi automatica di oligonucleotidi in fase liquida [Brevetto]

Apparecchiatura e processo per la sisntesi automatica di oligomeri in fase liquida in presenza di polimero di supporto solubile, in cui detta apparecchiatura comprende un'unità logica che consente la gestione automatica dell'alimentazione dosata dei reattivi e dei solventi, del tempo di permanenza della miscela all'interno del reattore e delle operazioni di purificazione

- Metodo per la predizione della resa finale ed il controllo automatico della sintesi di peptidi in fase solida [Brevetto]

Metodo per la predizione della resa finale ed il controllo automatico della sintesi di peptidi in fase solida comprendente la rilevazione del seganle conduttimetrico generato dalla miscela di reazione nei primi 5 minuti di reazione, il campionamento di detto segnale attraverso un sistema di conversione analogico/digitale e l'elaborazione dell'andamento del segnale mediante rete neurale

2018 - A gene expression signature of Retinoblastoma loss-of-function predicts resistance to neoadjuvant chemotherapy in ER-positive/HER2-positive breast cancer patients [Articolo su rivista]
Risi, Emanuela; Grilli, Andrea; Migliaccio, Ilenia; Biagioni, Chiara; Mccartney, Amelia; Guarducci, Cristina; Bonechi, Martina; Benelli, Matteo; Vitale, Stefania; Biganzoli, Laura; Bicciato, Silvio; Di Leo, Angelo; Malorni, Luca

HER2-positive (HER2+) breast cancers show heterogeneous response to chemotherapy, with the ER-positive (ER+) subgroup deriving less benefit. Loss of retinoblastoma tumor suppressor gene (RB1) function has been suggested as a cardinal feature of breast cancers that are more sensitive to chemotherapy and conversely resistant to CDK4/6 inhibitors. We performed a retrospective analysis exploring RBsig, a gene signature of RB loss, as a potential predictive marker of response to neoadjuvant chemotherapy in ER+/HER2+ breast cancer patients.We selected clinical trials of neoadjuvant chemotherapy +/- anti-HER2 therapy in HER2+ breast cancer patients with available information on gene expression data, hormone receptor status, and pathological complete response (pCR) rates. RBsig expression was computed in silico and correlated with pCR.Ten studies fulfilled the inclusion criteria and were included in the analysis (514 patients). Overall, of 211 ER+/HER2+ breast cancer patients, 49 achieved pCR (23%). The pCR rate following chemotherapy +/- anti-HER2 drugs in patients with RBsig low expression was significantly lower compared to patients with RBsig high expression (16% vs. 30%, respectively; Fisher's exact test p = 0.015). The area under the ROC curve (AUC) was 0.62 (p = 0.005). In the 303 ER-negative (ER-)/HER2+ patients treated with chemotherapy +/- anti-HER2 drugs, the pCR rate was 43%. No correlation was found between RBsig expression and pCR rate in this group.Low expression of RBsig identifies a subset of ER+/HER2+ patients with low pCR rates following neoadjuvant chemotherapy +/- anti-HER2 therapy. These patients may potentially be spared chemotherapy in favor of anti-HER2, endocrine therapy, and CDK 4/6 inhibitor combinations.

2018 - Bimodal CD40/Fas-Dependent Crosstalk between iNKT Cells and Tumor-Associated Macrophages Impairs Prostate Cancer Progression [Articolo su rivista]
Cortesi, Filippo; Delfanti, Gloria; Grilli, Andrea; Calcinotto, Arianna; Gorini, Francesca; Pucci, Ferdinando; Lucianò, Roberta; ` Grioni, Matteo; Recchia, Alessandra; Benigni, Fabio; Briganti, Alberto; Salonia, Andrea; De Palma, Michele; Bicciato, Silvio; Doglioni, Claudio; Bellone, Matteo; Casorati, Giulia; Dellabona, Paolo.

Heterotypic cellular and molecular interactions in the tumor microenvironment (TME) control cancer progression. Here, we show that CD1d-restricted invariant natural killer (iNKT) cells control prostate cancer (PCa) progression by sculpting the TME. In a mouse PCa model, iNKT cells restrained the proangiogenic and immunosuppressive capabilities of tumor-infiltrating immune cells by reducing proangiogenic TIE2+, M2-like macrophages (TEMs), and sustaining pro-inflammatory M1-like macrophages. iNKT cells directly contacted macrophages in the PCa stroma, and iNKT cell transfer into tumorbearing mice abated TEMs, delaying tumor progression. iNKT cells modulated macrophages through the cooperative engagement of CD1d, Fas, and CD40, which promoted selective killing of M2-like and survival of M1-like macrophages. Human PCa aggressiveness associate with reduced intra-tumoral iNKT cells, increased TEMs, and expression of pro-angiogenic genes, underscoring the clinical significance of this crosstalk. Therefore, iNKT cells may control PCa through mechanisms involving differential macrophage modulation, which may be harnessed for therapeutically reprogramming the TME.

2018 - Chromosome positioning in interphase nuclei of hematopoietic stem cell and myeloid precursor [Articolo su rivista]
Lomiento, Mariana; Mammoli, Fabiana; Mazza, Emilia Maria Cristina; Bicciato, Silvio; Ferrari, Sergio

Human myelopoiesis is an intriguing biological process during which multipotent stem cells limit their differentiation potential generating precursors that evolve into terminally differentiated cells. The differentiation process is correlated with differential gene expression and changes in nuclear architecture. In interphase, chromosomes are distinct entities known as chromosome territories and they show a radial localization that could result in a constrain of inter-homologous distance. This element plays a role in genome stability and gene expression. Here, we provide the first experimental evidence of 3D chromosomal arrangement considering two steps of human normal myelopoiesis. Specifically, multicolor 3D-FISH and 3D image analysis revealed that, in both normal human hematopoietic stem cells and myelod precursors CD14-, chromosomal position is correlated with gene density. However, we observed that inter-homologue distances are totally different during differentiation. This could be associated with differential gene expression that we found comparing the two cell types. Our results disclose an unprecedented framework relevant for deciphering the genomic mechanisms at the base of normal human myelopoiesis.

2018 - Computational methods for analyzing genome-wide chromosome conformation capture data [Articolo su rivista]
Nicoletti, Chiara; Forcato, Mattia; Bicciato, Silvio

In all organisms, chromatin is packed to fulfil structural constraints and functional requirements. The hierarchical model of chromatin organization in the 3D nuclear space encompasses different topologies at diverse scale lengths, with chromosomes occupying distinct volumes, further organized in compartments, inside which the chromatin fibers fold into large domains and short-range loops. In the recent years, the combination of chromosome conformation capture (3C) techniques and high-throughput sequencing allowed probing chromatin spatial organization at the whole genome-scale. 3C-based methods produce enormous amounts of genomic data that are analyzed using ad-hoc computational procedures. Here, we review the common pipelines and methods for the analysis of genome-wide chromosome conformation capture data, highlighting recent developments in key steps for the identification of chromatin structures.

2018 - Deficiency of immunoregulatory indoleamine 2,3-dioxygenase 1in juvenile diabetes [Articolo su rivista]
Orabona, Ciriana; Mondanelli, Giada; Pallotta, Maria T; Carvalho, Agostinho; Albini, Elisa; Fallarino, Francesca; Vacca, Carmine; Volpi, Claudia; Belladonna, Maria L; Berioli, Maria G; Ceccarini, Giulia; Esposito, Susanna Mr; Scattoni, Raffaella; Verrotti, Alberto; Ferretti, Alessandra; De Giorgi, Giovanni; Toni, Sonia; Cappa, Marco; Matteoli, Maria C; Bianchi, Roberta; Matino, Davide; Iacono, Alberta; Puccetti, Matteo; Cunha, Cristina; Bicciato, Silvio; Antognelli, Cinzia; Talesa, Vincenzo N; Chatenoud, Lucienne; Fuchs, Dietmar; Pilotte, Luc; Van den Eynde, Benoît; Lemos, Manuel C; Romani, Luigina; Puccetti, Paolo; Grohmann, Ursula

A defect in indoleamine 2,3-dioxygenase 1 (IDO1), which is responsible for immunoregulatory tryptophan catabolism, impairs development of immune tolerance to autoantigens in NOD mice, a model for human autoimmune type 1 diabetes (T1D). Whether IDO1 function is also defective in T1D is still unknown. We investigated IDO1 function in sera and peripheral blood mononuclear cells (PBMCs) from children with T1D and matched controls. These children were further included in a discovery study to identify SNPs in IDO1 that might modify the risk of T1D. T1D in children was characterized by a remarkable defect in IDO1 function. A common haplotype, associated with dysfunctional IDO1, increased the risk of developing T1D in the discovery and also confirmation studies. In T1D patients sharing such a common IDO1 haplotype, incubation of PBMCs in vitro with tocilizumab (TCZ) - an IL-6 receptor blocker - would, however, rescue IDO1 activity. In an experimental setting with diabetic NOD mice, TCZ was found to restore normoglycemia via IDO1-dependent mechanisms. Thus, functional SNPs of IDO1 are associated with defective tryptophan catabolism in human T1D, and maneuvers aimed at restoring IDO1 function would be therapeutically effective in at least a subgroup of T1D pediatric patients.

2018 - Differential proteomic profile of leukemic CD34+ progenitor cells from chronic myeloid leukemia patients [Articolo su rivista]
Ricciardi, Maria Rosaria; Salvestrini, Valentina; Licchetta, Roberto; Mirabilii, Simone; Forcato, Mattia; Gugliotta, Gabriele; Salati, Simona; Castagnetti, Fausto; Rosti, Gianantonio; Breccia, Massimo; Alimena, Giuliana; Manfredini, Rossella; Bicciato, Silvio; Lemoli, Roberto Massimo; Tafuri, Agostino

Chronic Myeloid Leukemia (CML) is a stem cell disease sustained by a rare population of quiescent cells which are to some extent resistant to tyrosine kinase inhibitors (TKIs). BCR-ABL oncogene activates multiple cross-talking signal transduction pathways (STP), such as RAS/MEK/ERK, PI3K/Akt, Wnt and STAT5, contributing to abnormal proliferation of clonal cells. From this perspective, the aim of this study was to analyze the expression and activation profile of STP involved in the mechanisms of cell proliferation/quiescence and survival of the progenitor CD34+ cells from chronic phase (CP) CML. Our results showed that CP-CML CD34+ progenitors were characterized by significant lower phosphorylation of proteins involved in the regulation of growth and cell survival, such as tyrosine kinases of the Src family and members of STAT family, and by a significant higher phosphorylation of p53 (Ser15), compared to normal CD34+ cells from healthy donors. Consistent with these results, cell cycle analysis demonstrated that CP-CML CD34+ cells were characterized by higher percentage of cells in G0-phase compared to normal CD34+ cells. Analysis of expression profile on proteins involved in the apoptotic machinery revealed that, in addition, CD34+ cells from CP-CML were characterized by a significant lower expression of catalase and higher expression of HSP27 and FADD. In sum, we report that CD34+ cells from CP-CML are characterized by a proteomic and phospho-proteomic profile that promotes quiescence through the inhibition of proliferation and the promotion of survival. This differential signaling activation network may be addressed by novel targeted therapies aimed at eradicating CML stem cells.

2018 - Dynamics of cellular states of fibro-adipogenic progenitors during myogenesis and muscular dystrophy [Articolo su rivista]
Malecova, Barbora; Gatto, Sole; Etxaniz, Usue; Passafaro, Magda; Cortez, Amy; Nicoletti, Chiara; Giordani, Lorenzo; Torcinaro, Alessio; De Bardi, Marco; Bicciato, Silvio; De Santa, Francesca; Madaro, Luca; Puri, Pier Lorenzo

Fibro-adipogenic progenitors (FAPs) are currently defined by their anatomical position, expression of non-specific membrane-associated proteins, and ability to adopt multiple lineages in vitro. Gene expression analysis at single-cell level reveals that FAPs undergo dynamic transitions through a spectrum of cell states that can be identified by differential expression levels of Tie2 and Vcam1. Different patterns of Vcam1-negative Tie2highor Tie2lowand Tie2low/Vcam1-expressing FAPs are detected during neonatal myogenesis, response to acute injury and Duchenne Muscular Dystrophy (DMD). RNA sequencing analysis identified cell state-specific transcriptional profiles that predict functional interactions with satellite and inflammatory cells. In particular, Vcam1-expressing FAPs, which exhibit a pro-fibrotic expression profile, are transiently activated by acute injury in concomitance with the inflammatory response. Aberrant persistence of Vcam1-expressing FAPs is detected in DMD muscles or upon macrophage depletion, and is associated with muscle fibrosis, thereby revealing how disruption of inflammation-regulated FAPs dynamics leads to a pathogenic outcome.

2018 - Enzymatic Inactivation of Oxysterols in Breast Tumor Cells Constraints Metastasis Formation by Reprogramming the Metastatic Lung Microenvironment [Articolo su rivista]
Moresco, Marta A; Raccosta, Laura; Corna, Gianfranca; Maggioni, Daniela; Soncini, Matias; Bicciato, Silvio; Doglioni, Claudio; Russo, Vincenzo

Recent evidence indicates that immune cells contribute to the formation of tumor metastases by regulating the pre-metastatic niche. Whether tumor-derived factors involved in primary tumor formation play a role in metastasis formation is poorly characterized. Oxysterols act as endogenous regulators of lipid metabolism through the interaction with the nuclear Liver X Receptors-(LXR)alpha and LXR beta. In the context of tumor development, they establish a pro-tumor environment by dampening antitumor immune responses, and by recruiting pro-angiogenic and immunosuppressive neutrophils. However, the ability of LXR/oxysterol axis to promote tumor invasion and metastasis by exploiting immune cells, is still up to debate. In this study we provide evidence that oxysterols participate in the primary growth of orthotopically implanted 4T1 breast tumors by establishing a tumor-promoting microenvironment. Furthermore, we show that oxysterols are involved in the metastatic spread of 4T1 breast tumors, since their enzymatic inactivation mediated by the sulfotransferase 2B1b, reduces the number of metastatic cells in the lungs of tumor-bearing mice. Finally, we provide evidence that oxysterols support the metastatic cascade by modifying the lung metastatic niche, particularly allowing the recruitment of tumor-promoting neutrophils. These results identify a possible new metastatic pathway to target in order to prevent metastasis formation in breast cancer patients.

2018 - GDA, a web-based tool for Genomics and Drugs integrated analysis [Articolo su rivista]
Caroli, Jimmy; Sorrentino, Giovanni; Forcato, Mattia; Del Sal, Giannino; Bicciato, Silvio

Several major screenings of genetic profiling and drug testing in cancer cell lines proved that the integration of genomic portraits and compound activities is effective in discovering new genetic markers of drug sensitivity and clinically relevant anticancer compounds. Despite most genetic and drug response data are publicly available, the availability of user-friendly tools for their integrative analysis remains limited, thus hampering an effective exploitation of this information. Here, we present GDA, a web-based tool for Genomics and Drugs integrated Analysis that combines drug response data for >50 800 compounds with mutations and gene expression profiles across 73 cancer cell lines. Genomic and pharmacological data are integrated through a modular architecture that allows users to identify compounds active towards cancer cell lines bearing a specific genomic background and, conversely, the mutational or transcriptional status of cells responding or not-responding to a specific compound. Results are presented through intuitive graphical representations and supplemented with information obtained from public repositories. As both personalized targeted therapies and drug-repurposing are gaining increasing attention, GDA represents a resource to formulate hypotheses on the interplay between genomic traits and drug response in cancer. GDA is freely available at

2018 - MYC-driven epigenetic reprogramming favors the onset of tumorigenesis by inducing a stem cell-like state [Articolo su rivista]
Poli, Vittoria; Fagnocchi, Luca; Fasciani, Alessandra; Cherubini, Alessandro; Mazzoleni, Stefania; Ferrillo, Sara; Miluzio, Annarita; Gaudioso, Gabriella; Vaira, Valentina; Turdo, Alice; Giaggianesi, Miriam; Chinnici, Aurora; Lipari, Elisa; Bicciato, Silvio; Bosari, Silvano; Todaro, Matilde; Zippo, Alessio

Breast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes are difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Overexpression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor-initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.

2018 - The early expansion of anergic NKG2Apos/CD56dim/CD16neg natural killer represents a therapeutic target in haploidentical hematopoietic stem cell transplantation [Articolo su rivista]
Roberto, Alessandra; Di Vito, Clara; Zaghi, Elisa; Mazza, Emilia Maria Cristina; Capucetti, Arianna; Calvi, Michela; Tentorio, Paolo; Zanon, Veronica; Sarina, Barbara; Mariotti, Jacopo; Bramanti, Stefania; Tenedini, Elena; Tagliafico, Enrico; Bicciato, Silvio; Santoro, Armando; Roederer, Mario; Marcenaro, Emanuela; Castagna, Luca; Lugli, Enrico; Mavilio, Domenico

Natural killer cells are the first lymphocyte population to reconsti-tute early after non-myeloablative and T cell-replete haploidenti-cal hematopoietic stem cell transplantation with post-transplant infusion of cyclophosphamide. The study herein characterizes the transient and predominant expansion starting from the second week following haploidentical hematopoietic stem cell transplantation of a donor-derived unconventional subset of NKp46neg-low/CD56dim/CD16neg natural killer cells expressing remarkably high levels of CD94/NKG2A. Both transcription and phenotypic profiles indicated that unconventional NKp46neg-low/CD56dim/CD16neg cells are a distinct natural killer cell subpopulation with features of late stage differentiation, yet retaining prolifera-tive capability and functional plasticity to generate conventional NKp46pos/CD56bright/CD16neg-low cells in response to interleukin-15 plus interleukin-18. While present at low frequency in healthy donors, unconventional NKp46neg-low/CD56dim/CD16neg cells are greatly expanded in the seven weeks following haploidentical hematopoietic stem cell transplantation, and express high levels of the activating receptors NKG2D and NKp30 as well as of the lytic granules Granzyme-B and Perforin. Nonetheless, NKp46neg-low/CD56dim/CD16neg cells displayed a markedly defective cytotoxicity that could be reversed by blocking the inhibitory receptor CD94/NKG2A. These data open new and important perspectives to better understand the ontogenesis/homeostasis of human natural killer cells and to develop a novel immune-therapeutic approach that targets the inhibitory NKG2A check-point, thus unleashing natural killer cell alloreactivity early after haploidentical hematopoietic stem cell transplantation.

2018 - Transcriptional addiction in cancer cells is mediated by YAP/TAZ through BRD4 [Articolo su rivista]
Zanconato, Francesca; Battilana, Giusy; Forcato, Mattia; Filippi, Letizia; Azzolin, Luca; Manfrin, Andrea; Quaranta, Erika; Di Biagio, Daniele; Sigismondo, Gianluca; Guzzardo, Vincenza; Lejeune, Pascale; Haendler, Bernard; Krijgsveld, Jeroen; Fassan, Matteo; Bicciato, Silvio; Cordenonsi, Michelangelo; Piccolo, Stefano

Cancer cells rely on dysregulated gene expression. This establishes specific transcriptional addictions that may be therapeutically exploited. Yet, the mechanisms that are ultimately responsible for these addictions are poorly understood. Here, we investigated the transcriptional dependencies of transformed cells to the transcription factors YAP and TAZ. YAP/TAZ physically engage the general coactivator bromodomain-containing protein 4 (BRD4), dictating the genome-wide association of BRD4 to chromatin. YAP/TAZ flag a large set of enhancers with super-enhancer-like functional properties. YAP/TAZ-bound enhancers mediate the recruitment of BRD4 and RNA polymerase II at YAP/TAZ-regulated promoters, boosting the expression of a host of growth-regulating genes. Treatment with small-molecule inhibitors of BRD4 blunts YAP/TAZ pro-tumorigenic activity in several cell or tissue contexts, causes the regression of pre-established, YAP/TAZ-addicted neoplastic lesions and reverts drug resistance. This work sheds light on essential mediators, mechanisms and genome-wide regulatory elements that are responsible for transcriptional addiction in cancer and lays the groundwork for a rational use of BET inhibitors according to YAP/TAZ biology.

2018 - Transcriptional profiling of human bronchial epithelial cell BEAS-2B exposed to diesel and biomass ultrafine particles [Articolo su rivista]
Grilli, Andrea; Bengalli, Rossella; Longhin, Eleonora; Capasso, Laura; Proverbio, Maria Carla; Forcato, Mattia; Bicciato, Silvio; Gualtieri, Maurizio; Battaglia, Cristina; Camatini, Marina

Background: Emissions from diesel vehicles and biomass burning are the principal sources of primary ultrafine particles (UFP). The exposure to UFP has been associated to cardiovascular and pulmonary diseases, including lung cancer. Although many aspects of the toxicology of ambient particulate matter (PM) have been unraveled, the molecular mechanisms activated in human cells by the exposure to UFP are still poorly understood. Here, we present an RNA-seq time-course experiment (five time point after single dose exposure) used to investigate the differential and temporal changes induced in the gene expression of human bronchial epithelial cells (BEAS-2B) by the exposure to UFP generated from diesel and biomass combustion. A combination of different bioinformatics tools (EdgeR, next-maSigPro and reactome FI app-Cytoscape and prioritization strategies) facilitated the analyses the temporal transcriptional pattern, functional gene set enrichment and gene networks related to cellular response to UFP particles.Results: The bioinformatics analysis of transcriptional data reveals that the two different UFP induce, since the earliest time points, different transcriptional dynamics resulting in the activation of specific genes. The functional enrichment of differentially expressed genes indicates that the exposure to diesel UFP induces the activation of genes involved in TNFa signaling via NF-kB and inflammatory response, and hypoxia. Conversely, the exposure to ultrafine particles from biomass determines less distinct modifications of the gene expression profiles. Diesel UFP exposure induces the secretion of biomarkers associated to inflammation (CCXL2, EPGN, GREM1, IL1A, IL1B, IL6, IL24, EREG, VEGF) and transcription factors (as NFE2L2, MAFF, HES1, FOSL1, TGIF1) relevant for cardiovascular and lung disease. By means of network reconstruction, four genes (STAT3, HIF1a, NFKB1, KRAS) have emerged as major regulators of transcriptional response of bronchial epithelial cells exposed to diesel exhaust.Conclusions: Overall, this work highlights modifications of the transcriptional landscape in human bronchial cells exposed to UFP and sheds new lights on possible mechanisms by means of which UFP acts as a carcinogen and harmful factor for human health.

2017 - A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells [Articolo su rivista]
Mondanelli, Giada; Bianchi, Roberta; Pallotta, Maria Teresa; Orabona, Ciriana; Albini, Elisa; Iacono, Alberta; Belladonna, Maria Laura; Vacca, Carmine; Fallarino, Francesca; Macchiarulo, Antonio; Ugel, Stefano; Bronte, Vincenzo; Gevi, Federica; Zolla, Lello; Verhaar, Auke; Peppelenbosch, Maikel; Mazza, Emilia Maria Cristina; Bicciato, Silvio; Laouar, Yasmina; Santambrogio, Laura; Puccetti, Paolo; Volpi, Claudia; Grohmann, Ursula

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.

2017 - Altered peritumoral microRNA expression predicts head and neck cancer patients with a high risk of recurrence [Articolo su rivista]
Ganci, Federica; Sacconi, Andrea; Manciocco, Valentina; Covello, Renato; Benevolo, Maria; Rollo, Francesca; Strano, Sabrina; Valsoni, Sara; Bicciato, Silvio; Spriano, Giuseppe; Muti, Paola; Fontemaggi, Giulia; Blandino, Giovanni

Head and neck squamous cell carcinoma is typically characterized by a high incidence of local recurrences. It has been extensively shown that mucosa from head and neck squamous cell carcinoma patients carries both genetic and gene expression alterations, which are mostly attributable to major etiologic agents of head and neck squamous cell carcinoma. We previously identified a signature of microRNAs (miRNAs) whose high expression in tumors is predictive of recurrence. Here, we investigated whether the deregulation of miRNA expression in the tumor-surrounding mucosa is correlated to disease recurrence. Specifically, comparing the miRNA expression in matched tumoral, peritumoral, and normal tissues collected from head and neck squamous cell carcinoma patients, we identified 35 miRNAs that are deregulated in both tumoral and peritumoral tissues as compared with normal matched samples. Four of these composed a miRNA signature that predicts head and neck squamous cell carcinoma local recurrence independently from prognostic clinical variables. The predictive power of the miRNA signature increased when using the expression levels derived from both the peritumoral and the tumoral tissues. The expression signal of the miRNAs composing the predictive signature correlated with the transcriptional levels of genes mostly associated with proliferation. Our results show that expression of miRNAs in tumor-surrounding mucosa may strongly contribute to the identification of head and neck squamous cell carcinoma patients at high risk of local recurrence.

2017 - Comparison of computational methods for Hi-C data analysis [Articolo su rivista]
Forcato, Mattia; Nicoletti, Chiara; Pal, Koustav; Livi, Carmen Maria; Ferrari, Francesco; Bicciato, Silvio

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.

2017 - Glucocorticoid receptor signalling activates YAP in breast cancer [Articolo su rivista]
Sorrentino, Giovanni; Ruggeri, Naomi; Zannini, Alessandro; Ingallina, Eleonora; Bertolio, Rebecca; Marotta, Carolina; Neri, Carmelo; Cappuzzello, Elisa; Forcato, Mattia; Rosato, Antonio; Mano, Miguel; Bicciato, Silvio; Del Sal, Giannino

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.

2017 - MCM7 and its hosted miR-25, 93 and 106b cluster elicit YAP/TAZ oncogenic activity in lung cancer [Articolo su rivista]
Lo Sardo, F; Forcato, Mattia; Sacconi, A; Capaci, V; Zanconato, F; di Agostino, S; Del Sal, G; Pandolfi, P. P; Strano, S; Bicciato, Silvio; Blandino, G.

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 behaviour 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 bi-oncogenic locus consisting of one gene and three hosted microRNAs.

2017 - Mechanical cues control mutant p53 stability through a mevalonate-RhoA axis [Articolo su rivista]
Ingallina, Eleonora; Sorrentino, Giovanni; Bertolio, Rebecca; Lisek, Kamil; Zannini, Alessandro; Azzolin, Luca; Severino, Luisa Ulloa; Scaini, Denis; Mano, Miguel; Mantovani, Fiamma; Rosato, Antonio; Bicciato, Silvio; Piccolo, Stefano; Del Sal, Giannino

Tumour-associated p53 missense mutants act as driver oncogenes affecting cancer progression, metastatic potential and drug resistance (gain-of-function)1. Mutant p53 protein stabilization is a prerequisite for gain-of-function manifestation; however, it does not represent an intrinsic property of p53 mutants, but rather requires secondary events2. Moreover, mutant p53 protein levels are often heterogeneous even within the same tumour, raising questions on the mechanisms that control local mutant p53 accumulation in some tumour cells but not in their neighbours2,3. By investigating the cellular pathways that induce protection of mutant p53 from ubiquitin-mediated proteolysis, we found that HDAC6/Hsp90-dependent mutant p53 accumulation is sustained by RhoA geranylgeranylation downstream of the mevalonate pathway, as well as by RhoA- and actin-dependent transduction of mechanical inputs, such as the stiffness of the extracellular environment. Our results provide evidence for an unpredicted layer of mutant p53 regulation that relies on metabolic and mechanical cues.

2017 - Regeneration of the entire human epidermis using transgenic stem cells [Articolo su rivista]
Hirsch, Tobias; Rothoeft, Tobias; Teig, Norbert; Bauer, Johann W.; Pellegrini, Graziella; De Rosa, Laura; Scaglione, Davide; Reichelt, Julia; Klausegger, Alfred; Kneisz, Daniela; Romano, Oriana; Seconetti, Alessia Secone; Contin, Roberta; Enzo, Elena; Jurman, Irena; Carulli, Sonia; Jacobsen, Frank; Luecke, Thomas; Lehnhardt, Marcus; Fischer, Meike; Kueckelhaus, Maximilian; Quaglino, Daniela; Morgante, Michele; Bicciato, Silvio; Bondanza, Sergio; De Luca, Michele

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, lifethreatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

2017 - The mutant p53-ID4 complex controls VEGFA isoforms by recruiting lncRNA MALAT1 [Articolo su rivista]
Pruszko, Magdalena; Milano, Elisa; Forcato, Mattia; Donzelli, Sara; Ganci, Federica; Di Agostino, Silvia; De Panfilis, Simone; Fazi, Francesco; Bates, David O; Bicciato, Silvio; Zylicz, Maciej; Zylicz, Alicja; Blandino, Giovanni; Fontemaggi, Giulia

The abundant, nuclear-retained, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been associated with a poorly differentiated and aggressive phenotype of mammary carcinomas. This long non-coding RNA (lncRNA) localizes to nuclear speckles, where it interacts with a subset of splicing factors and modulates their activity. In this study, we demonstrate that oncogenic splicing factor SRSF1 bridges MALAT1 to mutant p53 and ID4 proteins in breast cancer cells. Mutant p53 and ID4 delocalize MALAT1 from nuclear speckles and favor its association with chromatin. This enables aberrant recruitment of MALAT1 on VEGFA pre-mRNA and modulation of VEGFA isoforms expression. Interestingly, VEGFA-dependent expression signatures associate with ID4 expression specifically in basal-like breast cancers carrying TP53 mutations. Our results highlight a key role for MALAT1 in control of VEGFA isoforms expression in breast cancer cells expressing gain-of-function mutant p53 and ID4 proteins.

2017 - The proteasome inhibitor bortezomib controls indoleamine 2,3-dioxygenase 1 breakdown and restores immune regulation in autoimmune diabetes [Articolo su rivista]
Mondanelli, Giada; Albini, Elisa; Pallotta, Maria T.; Volpi, Claudia; Chatenoud, Lucienne; Kuhn, Chantal; Fallarino, Francesca; Matino, Davide; Belladonna, Maria L.; Bianchi, Roberta; Vacca, Carmine; Bicciato, Silvio; Boon, Louis; Ricci, Giovanni; Grohmann, Ursula; Puccetti, Paolo; Orabona, Ciriana

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.

2017 - WoPPER: Web server for Position Related data analysis of gene Expression in Prokaryotes [Articolo su rivista]
Puccio, Simone; Grillo, Giorgio; Licciulli, Flavio; Severgnini, Marco; Liuni, Sabino; Bicciato, Silvio; De Bellis, Gianluca; Ferrari, Francesco; Peano, Clelia

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:

2016 - A comparative transcriptomic analysis of astrocytes differentiation from human neural progenitor cells [Articolo su rivista]
Magistri, Marco; Khoury, Nathalie; Mazza, Emilia Maria Cristina; Velmeshev, Dmitry; Lee, Jae K.; Bicciato, Silvio; Tsoulfas, Pantelis; Faghihi, Mohammad Ali

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.

2016 - APTANI: a computational tool to select aptamers through sequence-structure motif analysis of HT-SELEX data [Articolo su rivista]
Caroli, J; Taccioli, C; De La Fuente, A; Serafini, P; Bicciato, S

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.

2016 - Aging: A portrait from gene expression profile in blood cells [Articolo su rivista]
Calabria, Elisa; Cristina Mazza, Emilia Maria; Dyar, Kenneth Allen; Pogliaghi, Silvia; Bruseghini, Paolo; Morandi, Carlo; Salvagno, Gian Luca; Gelati, Matteo; Guidi, Gian Cesare; Bicciato, Silvio; Schiaffino, Stefano; Schena, Federico; Capelli, Carlo

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.

2016 - Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells [Articolo su rivista]
Volpi, Claudia; Mondanelli, Giada; Pallotta, Maria T.; Vacca, Carmine; Iacono, Alberta; Gargaro, Marco; Albini, Elisa; Bianchi, Roberta; Belladonna, Maria L.; Celanire, Sylvain; Mordant, Céline; Heroux, Madeleine; Royer-Urios, Isabelle; Schneider, Manfred; Vitte, Pierre-Alain; Cacquevel, Mathias; Galibert, Laurent; Poli, Sonia-Maria; Solari, Aldo; Bicciato, Silvio; Calvitti, Mario; Antognelli, Cinzia; Puccetti, Paolo; Orabona, Ciriana; Fallarino, Francesca; Grohmann, Ursula

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.

2016 - Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation [Articolo su rivista]
Cavazza, Alessia; Miccio, Annarita; Romano, Oriana; Petiti, Luca; Malagoli Tagliazucchi, Guidantonio; Peano, Clelia; Severgnini, Marco; Rizzi, Ermanno; De Bellis, Gianluca; Bicciato, Silvio; Mavilio, Fulvio

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.

2016 - Human liver-resident CD56<sup>bright</sup>/CD16<sup>neg</sup> NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways [Articolo su rivista]
Hudspeth, Kelly; Donadon, Matteo; Cimino, Matteo; Pontarini, Elena; Tentorio, Paolo; Preti, Max; Hong, Michelle; Bertoletti, Antonio; Bicciato, Silvio; Invernizzi, Pietro; Lugli, Enrico; Torzilli, Guido; Eric Gershwin, M.; Mavilio, Domenico

RATIONALE: 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. OBJECTIVES: 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. FINDINGS: Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56bright lr-NK cells that localize within hepatic sinusoids. CD56bright 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 CCL3pos Kupffer cells, CXCL16pos endothelial cells and CCL5pos T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56bright NK cells that constitutively express CCR5 and CXCR6. CD56bright lr-NK cells co-exist with CD56dim conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56dim c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. CONCLUSION: 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 CD56bright lr-NK cells will require modification of hepatic CCR5 and CXCR6.

2016 - Induction of Expandable Tissue-Specific Stem/Progenitor Cells through Transient Expression of YAP/TAZ [Articolo su rivista]
Panciera, Tito; Azzolin, Luca; Fujimura, Atsushi; Di Biagio, Daniele; Frasson, Chiara; Bresolin, Silvia; Soligo, Sandra; Basso, Giuseppe; Bicciato, Silvio; Rosato, Antonio; Cordenonsi, Michelangelo; Piccolo, Stefano

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.

2016 - LncRNA profiling in early-stage chronic lymphocytic leukemia identifies transcriptional fingerprints with relevance in clinical outcome [Articolo su rivista]
Ronchetti, D.; Manzoni, M.; Agnelli, L.; Vinci, C.; Fabris, S.; Cutrona, G.; Matis, S.; Colombo, M.; Galletti, S.; Taiana, E.; Recchia, A.G.; Bossio, S.; Gentile, M.; Musolino, C.; Di Raimondo, F.; Grilli, A.; Bicciato, S.; Cortelezzi, A.; Tassone, P.; Morabito, F.; Ferrarini, M.; Neri, A.

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-lncRNAsignature 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.

2016 - MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity [Articolo su rivista]
Moretti, Irene; Ciciliot, Stefano; Dyar, Kenneth A.; Abraham, Reimar; Murgia, Marta; Agatea, Lisa; Akimoto, Takayuki; Bicciato, Silvio; Forcato, Mattia; Pierre, Philippe; Uhlenhaut, N. Henriette; Rigby, Peter W. J.; Carvajal, Jaime J.; Blaauw, Bert; Calabria, Elisa; Schiaffino, Stefano

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.

2016 - T Cell Cancer Therapy Requires CD40-CD40L Activation of Tumor Necrosis Factor and Inducible Nitric-Oxide-Synthase-Producing Dendritic Cells [Articolo su rivista]
Marigo, Ilaria; Zilio, Serena; Desantis, Giacomo; Mlecnik, Bernhard; Agnellini, Andrielly H R; Ugel, Stefano; Sasso, Maria Stella; Qualls, Joseph E; Kratochvill, Franz; Zanovello, Paola; Molon, Barbara; Ries, Carola H; Runza, Valeria; Hoves, Sabine; Bilocq, Amélie M; Bindea, Gabriela; Mazza, Emilia M C; Bicciato, Silvio; Galon, Jérôme; Murray, Peter J; Bronte, Vincenzo

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.

2016 - Transcriptional, epigenetic and retroviral signatures identify regulatory regions involved in hematopoietic lineage commitment [Articolo su rivista]
Romano, Oriana; Peano, Clelia; Tagliazucchi, Guidantonio Malagoli; Petiti, Luca; Poletti, Valentina; Cocchiarella, Fabienne; Rizzi, Ermanno; Severgnini, Marco; Cavazza, Alessia; Rossi, Claudia; Pagliaro, Pasqualepaolo; Ambrosi, Alessandro; Ferrari, Giuliana; Bicciato, Silvio; De Bellis, Gianluca; Mavilio, Fulvio; Miccio, Annarita

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.

2016 - YAP enhances the pro-proliferative transcriptional activity of mutant p53 proteins [Articolo su rivista]
Di Agostino, Silvia; Sorrentino, Giovanni; Ingallina, Eleonora; Valenti, Fabio; Ferraiuolo, Maria; Bicciato, Silvio; Piazza, Silvano; Strano, Sabrina; Del Sal, Giannino; Blandino, Giovanni

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.

2015 - A multifactorial ‘Consensus Signature’ by in silico analysis to predict response to neoadjuvant anthracycline-based chemotherapy in triple-negative breast cancer [Articolo su rivista]
Turner, Natalie; Forcato, Mattia; Nuzzo, Simona; Malorni, Luca; Bicciato, Silvio; Di Leo, Angelo

BACKGROUND: Owing to the complex processes required for anthracycline-induced cytotoxicity, a prospectively defined multifactorial Consensus Signature (ConSig) might improve prediction of anthracycline response in triple-negative breast cancer (TNBC) patients, whose only standard systemic treatment option is chemotherapy. AIMS: We aimed to construct and evaluate a multifactorial signature, comprising measures of each function required for anthracycline sensitivity in TNBC. METHODS: ConSigs were constructed based on five steps required for anthracycline function: drug penetration, nuclear topoisomerase IIα (topoIIα) protein location, increased topoIIα messenger RNA (mRNA) expression, apoptosis induction, and immune activation measured by, respectively, HIF1α or SHARP1 signature, LAPTM4B mRNA, topoIIα mRNA, Minimal Gene signature or YWHAZ mRNA, and STAT1 signature. TNBC patients treated with neoadjuvant anthracycline-based chemotherapy without taxane were identified from publicly available gene expression data derived with Affymetrix HG-U133 arrays (training set). In silico analyses of correlation between gene expression data and pathological complete response (pCR) were performed using receiver-operating characteristic curves. To determine anthracycline specificity, ConSigs were assessed in patients treated with anthracycline plus taxane. Specificity, sensitivity, positive and negative predictive value, and odds ratio (OR) were calculated for ConSigs. Analyses were repeated in two validation gene expression data sets derived using different microarray platforms. RESULTS: In the training set, 29 of 147 patients had pCR after anthracycline-based chemotherapy. Various combinations of components were evaluated, with the most powerful anthracycline response predictors being ConSig1: (STAT1+topoIIα mRNA +LAPTM4B) and ConSig2: (STAT1+topoIIα mRNA+HIF1α). ConSig1 demonstrated high negative predictive value (85%) and high OR for no pCR (3.18) and outperformed ConSig2 in validation sets for anthracycline specificity. CONCLUSIONS: With further validation, ConSig1 may help refine selection of TNBC patients for anthracycline chemotherapy.

2015 - Aerobic glycolysis tunes YAP/TAZ transcriptional activity [Articolo su rivista]
Enzo, Elena; Santinon, Giulia; Pocaterra, Arianna; Aragona, Mariaceleste; Bresolin, Silvia; Forcato, Mattia; Grifoni, Daniela; Pession, Annalisa; Zanconato, Francesca; Guzzo, Giulia; Bicciato, Silvio; Dupont, Sirio

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.

2015 - Generation of human memory stem T cells after haploidentical T-replete hematopoietic stem cell transplantation [Articolo su rivista]
Cieri, Nicoletta; Oliveira, Giacomo; Greco, Raffaella; Forcato, Mattia; Taccioli, Cristian; Cianciotti, Beatrice; Valtolina, Veronica; Noviello, Maddalena; Vago, Luca; Bondanza, Attilio; Lunghi, Francesca; Marktel, Sarah; Bellio, Laura; Bordignon, Claudio; Bicciato, Silvio; Peccatori, Jacopo; Ciceri, Fabio; Bonini, Chiara

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.

2015 - Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth [Articolo su rivista]
Zanconato, Francesca; Forcato, Mattia; Battilana, Giusy; Azzolin, Luca; Quaranta, Erika; Bodega, Beatrice; Rosato, Antonio; Bicciato, Silvio; Cordenonsi, Michelangelo; Piccolo, Stefano

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.

2015 - Genome-wide definition of promoter and enhancer usage during neural induction of human embryonic stem cells [Articolo su rivista]
Poletti, Valentina; Delli Carri, Alessia; Malagoli Tagliazucchi, Guidantonio; Faedo, Andrea; Petiti, Luca; Mazza, Emilia Maria Cristina; Peano, Clelia; De Bellis, Gianluca; Bicciato, Silvio; Miccio, Annarita; Cattaneo, Elena; Mavilio, Fulvio

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.

2015 - Integrative Analysis of Copy Number and Gene Expression Data Suggests Novel Pathogenetic Mechanisms in Primary Myelofibrosis [Abstract in Rivista]
Salati, Simona; Zini, Roberta; Nuzzo, Simona; Guglielmelli, Paola; Pennucci, Valentina; Prudente, Zelia; Ruberti, Samantha; Rontauroli, Sebastiano; Norfo, Ruggiero; Bianchi, Elisa; Bogani, Costanza; Rotunno, Giada; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Salmoiraghi, Silvia; Pietra, Daniela; Ferrari, Sergio; Barosi, Giovanni; Rambaldi, Alessandro; Cazzola, Mario; Bicciato, Silvio; Tagliafico, Enrico; Vannucchi, Alessandro M.; Manfredini, Rossella

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, we integrated gene expression and copy number (CN) signals and identified several genomic abnormalities leading to a concordant alteration in gene expression levels. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus is accompanied by a coordinated transcriptional up-regulation in PMF patients. To assess the impact of PAOX deregulation on the survival of PMF and normal primary hematopoietic cells, PMF and normal donors cells were incubated with increasing doses of the PAOX inhibitor MDL-72,572. PAOX inhibition resulted in rapid cell death of PMF progenitor cells (5,7±0,9% of late apoptotic cells in NT sample vs 20,1±3,2% in 150μM treated-sample, p<0.05), while survival of normal CD34+ cells was not affected (0,97±0,09 of late apoptotic cells in NT sample vs 2,7±0,4 in 150μM treated-sample, p<0.05), as monitored by Annexin V/PI staining. These data suggest that PAOX inhibition could represent a therapeutic strategy to selectively target PMF cells without affecting normal hematopoietic cells' survival. Moreover, our integrative analysis of gene expression and CN data pointed out the concomitant copy number loss and transcriptional down-regulation in PMF patients of the chromatin modifier HMGXB4. Interestingly, silencing of HMGXB4 in CD34+ stem/progenitor cells induced megakaryocyte differentiation, as demonstrated by increased expression of CD41 (36±1,9% vs 26,1±2,9% at day 12, 52,5±4,9% vs 33±1% at day 14 of serum free liquid culture, p<0.05) and increased number of CFU-MK colonies (27,7±2,5 vs 18,1±1,3% in small CFU-MK colonies, p<0.05). On the other hand, HMGXB4 silencing repressed the erythroid differentiation, as indicated by a decrease in the percentage of cells positive for the erythroid marker GPA (10.8%±5.6 vs 24.8%±5, p<0.05) and significant decrease in Burst Forming Unit-Erythroid (BFU-E) and Colony-Forming Unit-Erythroid (CFU-E) (47,9±6 vs 60,5±7,6, p<0.05). Taken together, these data suggest that HMGXB4 silencing in human HSPCs favors megakaryocyte differentiation while restraining the erythroid lineage. 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 characterize PMF patients. In conclusion, our work sheds light on the influence of genomic abnormalities on gene expression regulation in PMF CD34+ cells and on their impact to features typical of PMF, such as a hyperplastic megakaryopoiesis and resistance to apoptosis, and therefore potentially contributing to the development of the myeloproliferative disease.

2015 - Integrative analysis of copy number and gene expression data suggests novel pathogenetic mechanisms in Primary Myelofibrosis [Articolo su rivista]
Salati, Simona; Zini, Roberta; Nuzzo, Simona; Guglielmelli, Paola; Pennucci, Valentina; Prudente, Zelia; Ruberti, Samantha; Rontauroli, Sebastiano; Norfo, Ruggiero; Bianchi, Elisa; Bogani, Costanza; Rotunno, Giada; Fanelli, Tiziana; Mannarelli, Carmela; Rosti, Vittorio; Salmoiraghi, Silvia; Pietra, Daniela; Ferrari, Sergio; Barosi, Giovanni; Rambaldi, Alessandro; Cazzola, Mario; Bicciato, Silvio; Tagliafico, Enrico; Vannucchi, Alessandro M.; Manfredini, Rossella

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, we integrated gene expression and copy number signals and identified several genomic abnormalities leading to a concordant alteration in gene expression levels. In particular, copy number gain in the polyamine oxidase (PAOX) gene locus is 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 characterize PMF patients. This article is protected by copyright. All rights reserved.

2015 - 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 [Articolo su rivista]
Taccioli, Cristian; Sorrentino, Giovanni; Zannini, Alessandro; Caroli, Jimmy; Beneventano, Domenico; Anderlucci, Laura; Lolli, Marco; Bicciato, Silvio; Del Sal, Giannino

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,, 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.

2015 - Notch is a direct negative regulator of the DNA-damage response [Articolo su rivista]
Vermezovic, Jelena; Adamowicz, Marek; Santarpia, Libero; Rustighi, Alessandra; Forcato, Mattia; Lucano, Caterina; Massimiliano, Lucia; Costanzo, Vincenzo; Bicciato, Silvio; Del Sal, Giannino; D'Adda Di Fagagna, Fabrizio

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.

2015 - 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 [Articolo su rivista]
Guarneri, Valentina; Dieci, Maria Vittoria; Frassoldati, Antonio; Maiorana, Antonino; Ficarra, Guido; Bettelli, Stefania; Tagliafico, Enrico; Bicciato, Silvio; Generali, Daniele Giulio; Cagossi, Katia; Bisagni, Giancarlo; Sarti, Samanta; Musolino, Antonino; Ellis, Catherine; Crescenzo, Rocco; Conte, Pierfranco

Background. The CHER-LOB randomized phase II study showed that the combination of lapatinib and trastuzumab plus chemotherapy increases the pathologic complete re- mission (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 trastu- zumab, 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. Results. 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 5.323). For patients receiving trastuzumab plus lapatinib, the probabil- ity of pCR was higher in PIK3CA wild-type tumors (48.4% vs. 12.5%; p 5.06). 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. Conclusion. 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.

2015 - The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle [Articolo su rivista]
Dyar, Kenneth A.; Ciciliot, Stefano; Tagliazucchi Malagoli, Guidantonio; Pallafacchina, Giorgia; Tothova, Jana; Argentini, Carla; Agatea, Lisa; Abraham, Reimar; Ahdesmäki, Miika; Forcato, Mattia; Bicciato, Silvio; Schiaffino, Stefano; Blaauw, Bert

OBJECTIVE: 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. METHODS: 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. RESULTS: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. CONCLUSIONS: 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 Ca2+-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.

2015 - The tissue inhibitor of metalloproteinases 1 increases the clonogenic efficiency of human hematopoietic progenitor cells through CD63/PI3K/Akt signaling [Articolo su rivista]
Rossi, Lara; Forte, Dorian; Migliardi, Giorgia; Salvestrini, Valentina; Buzzi, Marina; Ricciardi, Maria Rosaria; Licchetta, Roberto; Tafuri, Agostino; Bicciato, Silvio; Cavo, Michele; Catani, Lucia; Lemoli, Roberto M.; Curti, Antonio

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.

2014 - Aryl hydrocarbon receptor control of a disease tolerance defence pathway [Articolo su rivista]
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.

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.

2014 - Characterization of a genetic mouse model of lung cancer: a promise to identify Non-Small Cell Lung Cancer therapeutic targets and biomarkers. [Articolo su rivista]
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

BACKGROUND: 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. RESULTS: We performed RNA-seq profiling on total RNA extracted from lungs of a 30 week-old K-rasLA1/p53R172HΔg 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-rasLA1/p53R172HΔg 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-rasLA1/p53R172HΔg 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. CONCLUSIONS: Our study provided a complete transcription overview of the K-rasLA1/p53R172HΔg mouse NSCLC model. This approach allowed the detection of ADORA3 as a potential target for antibody-based therapy in p53 mutated tumors.

2014 - Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering [Articolo su rivista]
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.

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.

2014 - Gene expression profiling of human fibrocytic myeloid-derived suppressor cells (f-MDSCs) [Articolo su rivista]
Mazza, Emilia Maria Cristina; Zoso, Alessia; Mandruzzato, Susanna; Bronte, Vincenzo; Serafini, Paolo; Inverardi, Luca; Bicciato, Silvio

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

2014 - Human fibrocytic myeloid-derived suppressor cells express IDO and promote tolerance via Treg-cell expansion [Articolo su rivista]
Zoso, Alessia; Mazza, Emilia M C; Bicciato, Silvio; Mandruzzato, Susanna; Bronte, Vincenzo; Serafini, Paolo; Inverardi, Luca

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.

2014 - MafB is a downstream target of the IL-10/STAT3 signaling pathway, involved in the regulation of macrophage de-activation. [Articolo su rivista]
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

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.

2014 - Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock. [Articolo su rivista]
Dyar, Ka; Ciciliot, S; Wright, Le; Biensø, Rs; Tagliazucchi, Gm; Patel, Vr; Forcato, Mattia; Paz, Mi; Gudiksen, A; Solagna, F; Albiero, M; Moretti, I; Eckel Mahan, Kl; Baldi, P; Sassone Corsi, P; Rizzuto, R; Bicciato, Silvio; Pilegaard, H; Blaauw, B; Schiaffino, S.

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

2014 - Mutant p53 Reprograms TNF Signaling in Cancer Cells through Interaction with the Tumor Suppressor DAB2IP [Articolo su rivista]
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.

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.

2014 - Prolyl-isomerase Pin1 controls normal and cancer stem cells of the breast. [Articolo su rivista]
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

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

2014 - Quantitative phenotypic analysis of multistress response in Zygosaccharomyces rouxii complex. [Articolo su rivista]
Solieri L;C Dakal T;Bicciato S

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 732T and Z. sapae strains ABT301T 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 ABT301T 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

2014 - Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro. [Articolo su rivista]
Italiani P;Mazza EM;Lucchesi D;Cifola I;Gemelli C;Grande A;Battaglia C;Bicciato S;Boraschi D

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

2014 - UCbase 2.0: ultraconserved sequences database (2014 update) [Articolo su rivista]
Lomonaco, V; Martoglia, Riccardo; Mandreoli, Federica; Anderlucci, L; Emmett, W; Bicciato, Silvio; Taccioli, C.

UCbase 2.0 ( 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:

2014 - YAP/TAZ Incorporation in the β-Catenin Destruction Complex Orchestrates the Wnt Response. [Articolo su rivista]
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

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

2013 - Cancer gene prioritization by integrative analysis of mRNA expression and DNA copy number data: a comparative review [Articolo su rivista]
Lahti L; Schäfer M; Klein HU; Bicciato S; Dugas M.

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

2013 - IL-7 and IL-15 instruct the generation of human memory stem T cells from naïve precursors [Articolo su rivista]
N. Cieri; B. Camisa; F. Cocchiarella; M. Forcato; G.Oliveira; E. Provasi; A. Bondanza; C. Bordignon; J. Peccatori; F.Ciceri; M.T.Lupo-Stanghellini; F. Mavilio; A. Mondino; S. Bicciato; A. Recchia; C. Bonini

Long-living memory stem T cells (TSCM) 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+ TSCM lymphocytes starting from naïve 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 TSCM 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 naïve and central memory cells. When transplanted in immunodeficient mice, gene-modified naïve-derived TSCM 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 TSCM are the only T-cell subset able to expand and mediate GvHD upon serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for TSCM generation and pave the way for their clinical rapid exploitation in adoptive cell therapy


The model grass Brachypodium distachyon L (Brachypodium) has recently revealed its potential for studying grass-pathogen interactions. In particular, the identification of genomic regions associated with resistance to the false brome rust fungus Puccinia brachypodii offered perspectives to elucidate the genetic and molecular basis of this trait. In this study, we aimed to: 1) provide an initial whole-genome expression dataset for Brachypodium-P. brachypodii interaction in the two inbred lines Bd3-1 (resistant) and Bd1-1 (susceptible), and 2) fine mapping and cloning Rpbq2 and Rpbq3: to increase the resolution of QTL mapping and to reduce the number of candidate genes underlying QTL LOD curves. For the first, aim the two inbred lines have been characterized macroscopically and by confocal microscopy to follow the development of the fungus and the formation of rust infection structures. The expression of six brachypodium genes, homologous to known wheat and barley defence-related genes, was monitored by qRT-PCR analysis in Bd3-1 and Bd1-1 at three time points (18, 24 and 72 hours post infection, hpi). The 18 hpi time point was selected for transcriptome profiling on the basis of the expression profiles of the defence genes. The Affymetrix Brachypodium Tiling Array (BradiAR1b520742) revealed that expression levels of a set of genes (more than 100 in total) were altered in infected plants, mainly in the resistant line Bd3-1. At 18 hpi a significant re-programming of host metabolism occurred in infected leaves, with a modulation of genes involved in different metabolic networks such as defence, glycolysis, aminoacid and nitrogen metabolism. This study represents the first characterization of the functional genomic basis of resistance to a rust species in the model plant Brachypodium, and could be useful for translational genomics to ‘complex’ cereals. For the second aim, fine mapping Rpbq2 and Rpbq3, a new large segregating RIL population has been developed for each QTL separately. Selection of Bd3-1 x Bd1-1 RILs heterozygous for the QTLs has been completed based on flanking marker haplotypes, with the target QTL in a heterozygous state, while the other two QTLs were selected to be homozygous for the susceptible allele. These marker-selected heterozygous RILs have been selfed to obtain large segregating populations for each QTL. These results represent the first steps of a genetic approach towards the cloning of Rpbq2 and Rpbq3 determinants, and for their possible exploitation in cereals.

2012 - Comparative genomics and transcriptional profiles of Saccharopolyspora erythraea NRRL 2338 and a classically improved erythromycin over-producing strain [Articolo su rivista]
Peano C; Talà A; Corti G; Pasanisi D; Durante M; Mita G; Bicciato S; De Bellis G; Alifano P.

BACKGROUND: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.RESULTS: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.CONCLUSIONS:This study helps to clarify the mechanisms underlying antibiotic overproduction providing valuable information about new possible molecular targets for rationale strain improvement.

2012 - Hmgb3 Is Regulated by MicroRNA-206 during Muscle Regeneration [Articolo su rivista]
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

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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

GROHMANN U.; Bicciato S.; Bronte V.; Allavena P

We plan, in particular, to study Arg1 and IDO and their interaction with the tumor microenvironment both in human and experimental settings, so as to define their mutual influence and develop nanotechnology- based bioactive molecules specifically targeting the enzyme-related functions. Thus the project is meant (i) to be nationally/internationally oriented exploiting domestic and European networks; (ii) to represent a multidisciplinary effort; (iii) to have broader biological significance because both IDO and arginase are general regulators of immunity (including human retroviral infections); (iv) to deal with scientific issues, including IDO and arginase, that have recently been attracting much interest worldwide.

2012 - Role of TAZ as Mediator of Wnt Signaling [Articolo su rivista]
Azzolin L; Zanconato F; Bresolin S; Forcato M; Basso G; Bicciato S; Cordenonsi M; Piccolo S

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.

2012 - SHARP1 suppresses breast cancer metastasis by promoting degradation of hypoxia-inducible factors [Articolo su rivista]
Montagner, M; Enzo, E; Forcato, M; Zanconato, F; Parenti, A; Rampazzo, E; Basso, G; Leo, G; Rosato, A; Bicciato, S; Cordenonsi, M; Piccolo, S.

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.

2011 - Indoleamine 2,3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells [Articolo su rivista]
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.

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

2011 - Microarray Data Analysis: Gene Regulatory Networks [Capitolo/Saggio]
Bellazzi R; Bicciato S; Cobelli C; Di Camillo B; Ferrazzi F; Magni P; Sacchi L; Toffolo G

Cellular processes involve million of molecules playing a coherent role in the exchange of matter, energy and information both among themselves and with the environment. These processes are regulated by proteins, whose expression is controlled by a tight network of interactions between genes, proteins and other molecules. There is evidence that some pathologies of major social impact, like cancer and diabetes, involves groups of genes and proteins functionally related (pathways) rather than the expression of a single gene or protein. Therefore, it is important to investigate the global modifications of a specific regulatory pathway rather than the expression of a single gene. This is a major goal of systems biology approaches, devoted to the elucidation of the complex network of interacting DNAs sequences, RNAs and proteins regulating and controlling gene expression. Today, high-throughput technologies such as microarray and mass spectrometry, measure the cellular molecular expression in a given instant, thus making possible, at least in principle, the reconstruction of the regulatory network from its observed output through reverse engineering approaches. Unfortunately, microarray technology cost restricts the number of samples (order of 101-102) available for each experiment with respect to the number of monitored genes (order of 104). Mass spectrometry techniques have some limitations as well, since at present they are not able to provide a precise quantification of protein expression and require to identify the original protein from the spectrum of its fragments by mining specific databases. For these reasons, reverse engineering approaches are usually limited to very general and abstract models, where RNA expression is considered as a proxy of protein expression in controlling gene transcription. Several reverse engineering approaches based on this model have been proposed in the last years to infer gene regulatory networks from microarray gene expression data. Among them Boolean models, models based on differential equations, Bayesian networks and methods based on pair-wise gene expression correlation.

2011 - Microarray Data Analysis: General Concepts, Gene Selection, and Classification [Capitolo/Saggio]
Bellazzi R; Bicciato S; Cobelli C; Di Camillo B; Ferrazzi F; Magni P; Sacchi L; Toffolo G

Disclosures from the genome sequencing projects facilitated the development of novel techniques able to screen thousands of molecules in parallel and identify sets of potentially interesting sequences associated with physiological/pathological conditions. As a consequence, high-throughput, large-scale experimental methodologies, combined with bioinformatics analysis of DNA, RNA, and protein data, projected biological sciences into the so-called post-genomic-functional genomics era. The exploration of all genes or proteins at once, in a systematic fashion, represents a sort of revolution, shifting molecular biology and medicine research from a reductionistic, hypothesis-driven approach towards deciphering how genes and their products work, how they interact in pathways within the cells, and what roles they play in health and disease [1-2]. Oligonucleotide and cDNA microarrays for transcriptional profiling (Lockhart et al. 1996, Schena et al. 1995) allow measuring such interaction patterns, thus representing an unprecedented opportunity to boost the identification of diagnostic and therapeutic targets (Brown et al. 1999).The principle of a microarray for gene expression analysis is basically that of the classical northern-blot extended to the whole genome level. Specifically, mRNA from a given cell line or tissue is labeled with a fluorescent dye and hybridized to a large number of DNA sequences, immobilized on a solid surface (i.e., a glass slide or a silica wafer) in an ordered array. In such a way, tens of thousands of transcript species can be detected simultaneously, exploiting the highly specific complementary of nucleic acids. Indeed, mRNA molecules (targets) will couple with the corresponding complementary probe immobilized on the array and the fluorescence emission of any spot in the array will represent a quantification of the expression level of each target.

2011 - Molecular Portrait of Clear Cell Renal Cell Carcinoma: An Integrative Analysis of Gene Expression and Genomic Copy Number Profiling [Capitolo/Saggio]
Battaglia C.; Mangano E.; Bicciato S.; Frascati F.; Nuzzo S.; Tinaglia V.; Bianchi C.; Perego RA.; Cifola I

Renal cell carcinoma (RCC) incidence accounts for about 3 to 10 cases per 100,000 individuals with a predilection for adult males over 60 year old (1.6:1 male/female ratio) (Chow, 2010; Nese, 2009). In Europe, about 60,000 individuals are affected by RCC every year, with a mortality rate of about 18,000 subjects and an incidence rate for all stages steadily rising over the last three decades. Although inherited forms occur in a number of familial cancer syndromes, as the well-known von Hippel-Lindau (VHL) syndrome, RCC is commonly sporadic (Cohen & McGovern, 2005; Kaelin, 2007) and, as recently highlighted by the National Cancer Institute (NCI), influenced by the interplay between exposure to environmental risk factors and genetic susceptibility of exposed individuals (Chow et al., 2010). Being poorly symptomatic in early phases, many cases become clinically detectable only when already advanced and, as such, therapy-resistant (Motzer, 2011). Based on histology, RCC can be classified into several subtypes, i.e., clear cell (80% of cases), papillary (10%), chromophobe (5%) and oncocytoma (5%), each one characterized by specific histo- pathological features, malignant potential and clinical outcome (Cohen & McGovern, 2005). Patient stratification is normally achieved using prognostic algorithms and nomograms based on multiple clinico-pathological factors such as TNM stage, Fuhrman nuclear grade, tumor size, performance status, necrosis and other hematological indices (Flanigan et al., 2011), although the most efficient predictors of survival and recurrence are based on nuclear grade alone (Nese et al., 2009). As recently reviewed by Brannon et al. (Brannon & Rathmell, 2010), a finer RCC subtype classification could be obtained exploiting the vast amount of genomic and transcriptional data that have been presented in numerous studies. For instance, several authors proposed a molecular classification of RCC based on differential gene expression profiles, with any subtype characterized by the activation of distinct gene sets (Brannon, 2010; Furge, 2004; Skubitz, 2006; Sültmann, 2005; Zhang, 2008), while others identified RCC-specific biomarkers (e.g. CA9, ki67, VEGF proteins, phosphorylated AKT, PTEN, HIF-1). Lately, it has been reported that microRNAs, a small class of non coding RNA molecules, could contribute to RCC development at different levels and may represent a new group of potential tumor biomarkers (Redova et al., 2011). Despite the numerous efforts in dissecting the molecular features of RCC through functional genomics, not a single transcriptional signature or biomarker has gained approval for clinical application yet (Arsanious, 2009; Eichelberg, 2009; Lam, 2007; Yin-Goen, 2006), so that the identification of novel molecular markers to improve early diagnosis and prognostic prediction and of candidate targets to develop new therapeutic approaches remains of primary importance for this pathology.

2011 - Molecular basis for triple negative breast cancer metastasis: new tools for diagnosis and therapy [Partecipazione a progetti di ricerca]
Bicciato S; Di Leo A; D'Incalci M; Maestro R; Piccolo S; Rizzuto R; Rosato A; Schneider C; Veronesi A

Breast cancer is a heterogeneous disease, both biologically and clinically. Triple Negative Breast Cancer (TNBC) accounts for 20% of all breast cancers. Despite being the most aggressive breast cancer subtype, there is still no standard clinical therapy to curtail the innate aggressive behavior, marked by high rates of relapse, visceral metastases and early death (1). Urgent clinical priorities for TNBC patients are tools for identification of high likelihood of metastatic disease and development of targeted use of therapies to prevent metastases.Lack of therapeutic approaches to combat TNBC greatly rests on incomplete understanding of the mechanisms promoting tumor aggressiveness and the invasion-metastasis cascade. Recently, work from our groups provided key advances in this field by identifying two novel molecular axes promoting breast cancer aggressiveness and metastasis, namely the mutant-p53/p63/Pin1 axis and the microRNA-103-107/Dicer axis. These results provide an unprecedented blend of new molecules and mechanisms, with proven functional relevance in animal models for breast cancer metastasis, and with profound implications for both diagnosis and treatment of metastatic cancers in humans. We propose here to build on this knowledge to elaborate new diagnostic/prognostic tools and novel therapeutic strategies to manage TNBC, including the characterization and application of RNA- based therapeutics to inhibit pro-metastatic microRNAs.To tackle these objectives, we assembled a team of complementary nature, including basic researchers to identify, validate and develop novel molecular approaches, a pharmacology team with established expertise in drug development/validation and in clinical trials, and practicing medical oncologists providing an interface with breast cancer patients. Such collaboration provides a tremendous platform for embarking on the proposal outlined.

2011 - PREDA: an R-package to identify regional variations in genomic data [Articolo su rivista]
Ferrari F; Solari A; Battaglia C; Bicciato S.

SUMMARY: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.AVAILABILITY:PREDA is available in Bioconductor and at

2011 - Role of YAP/TAZ in mechanotransduction [Articolo su rivista]
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

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

2011 - The Hippo Transducer TAZ Confers Cancer Stem Cell-Related Traits on Breast Cancer Cells [Articolo su rivista]
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.

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.

2011 - The reconstruction of transcriptional networks reveals critical genes with implications for clinical outcome of multiple myeloma [Articolo su rivista]
Agnelli L; Forcato M; Ferrari F; Tuana G; Todoerti K; Walker BA; Morgan GJ; Lombardi L; Bicciato S; Neri A.

PURPOSE: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.EXPERIMENTAL DESIGN: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.RESULTS: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.CONCLUSIONS: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.

2010 - A MicroRNA targeting dicer for metastasis control [Articolo su rivista]
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.

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

2010 - Critical analysis of transcriptional and post-transcriptional regulatory networks in multiple myeloma [Articolo su rivista]
Biasiolo M; Forcato M; Possamai L; Ferrari F; Agnelli L; Lionetti M; Todoerti K; Neri A; Marchiori M; Bortoluzzi S; Bicciato S

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

2010 - HGM 2010 Programme / Abstract [Abstract in Rivista]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, Francesco; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors effort that, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning-on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNAs profiles from human CD34+ hematopoietic progenitor cells and in-vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblasts precursors, that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically up-regulated in one single cell progeny and their putative target genes, which resulted down-regulated. Among the up-regulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitors fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates the regulation of hematopoietic progenitors fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation.

2010 - Impact of probe annotation on the integration of miRNA-mRNA expression profiles for miRNA target detection [Articolo su rivista]
Sales G; Coppe A; Bicciato S; Bortoluzzi S; Romualdi C

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

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoieis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment [Abstract in Atti di Convegno]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, F; Orlandi, C; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors effort that, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning-on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNAs profiles from human CD34+ hematopoietic progenitor cells and in-vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblasts precursors, that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically up-regulated in one single cell progeny and their putative target genes, which resulted down-regulated. Among the up-regulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitors fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates the regulation of hematopoietic progenitors fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation.

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoieis: role of hsa-miR-299-5p in CD34+ progenitor cells commitment [Abstract in Atti di Convegno]
Tenedini, Elena; Roncaglia, Enrica; Ferrari, Francesco; Orlandi, Claudia; Bianchi, Elisa; Bicciato, Silvio; Tagliafico, Enrico; Ferrari, Sergio

Cell fate decisions in the hematopoietic system appear to be directed by an antagonistic or synergistic interplay of transcription factors that pivot immature blood progenitors for cell specification. Multipotent progenitors initially trigger a promiscuous transcriptional program and, as soon as they commit to a restricted fate, they reinforce unilineage gene expression and withdraw transcripts affiliated with alternative blood cell types. MicroRNAs appear to be especially pertinent in driving this particular behavior representing a new component of the hematopoietic gene regulatory network. In fact, the archetypal microRNA can potentially regulate hundreds of genes even if most targets contain isolated microRNA recognition sites that may be inadequate for complete gene silencing. According to Bartel’s theory, microRNAs mediated post-transcriptional control offers a more flexible and rapid way of tuning genes compared to transcriptional control (Bartel DP and Chen CZ, Nat Rev Genet 2004). These issues encouraged some investigators to explore the association of microRNAs and genes expression profiles obtained from the same cell type and advocated that microRNAs evolved to regulate gene expression programs and remove gene products unnecessary or potentially dangerous more rapidly than might occur by natural decay. Although many studies addressed the role of microRNAs during the normal myeloid differentiation process, only Georgantas and co-workers focused onto the impact of microRNAs on mRNA expression levels but limited the analyses to data obtained from human CD34+ stem/progenitor cells (Georgantas RW 3rd et al, PNAS 2007). In order to shed light onto the interplay of mRNAs and microRNAs during the normal myeloid commitment and verify that increased expression of a microRNA is skillful to modulate the levels of corresponding target mRNAs, we obtained microRNAs profiles from CD34+ hematopoietic progenitor cells (CD34 HPCs) and in-vitro differentiated precursors: erythroblasts, megakaryoblasts, monoblasts and myeloblasts (ERY, MKC, MONO and MYELO). We therefore analyzed these microRNA expression profiles together with the gene expression profiles of the same populations and observed that for the most part of the microRNAs specifically up-regulated in one single progeny an inverse correlation between microRNAs and down-regulated putative targets expression levels occurs, i.e. down-regulated genes showed an enrichment for the conserved putative targets of up-regulated microRNA. Among these microRNAs, hsa-miR-299-5p emerged as an interesting candidate to demonstrate how the integrated analysis of microRNA and mRNA expression data can help shedding light on the regulatory mechanisms governing cell differentiation. In particular, we used hsa-miR-299-5p to prove that the forced expression of a single lineage-specific microRNA is able to control the cell fate of CD34 HPCs grown in multilineage culture conditions. Clonogenic and liquid culture differentiation assays after gain- and loss-of-function experiments revealed that indeed hsa-miR-299-5p regulates hematopoietic progenitors fate modulating megakaryocytic-granulocytic versus erythroid-monocytic development.

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment [Articolo su rivista]
Tenedini E; Roncaglia E; Ferrari F; Orlandi C; Bianchi E; Bicciato S; Tagliafico E; Ferrari S.

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort, which, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA–mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes, which resulted in downregulation. Among the upregulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation.

2010 - Integrated analysis of microRNA and mRNA expression profiles in physiological myelopoiesis: role of hsa-mir-299-5p in CD34+ progenitor cells commitment [Articolo su rivista]
Tenedini E; Roncaglia E; Ferrari F; Orlandi C; Bianchi E; Bicciato S; Tagliafico E; Ferrari S.

Hematopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors' effort, which, in concert with microRNAs, drives cell fate and responds to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. We obtained microRNA profiles from human CD34+ hematopoietic progenitor cells and in vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblast precursors that we analyzed together with their gene expression profiles. The integrated analysis of microRNA-mRNA expression levels highlighted an inverse correlation between microRNAs specifically upregulated in one single-cell progeny and their putative target genes, which resulted in downregulation. Among the upregulated lineage-enriched microRNAs, hsa-miR-299-5p emerged as having a role in controlling CD34+ progenitor fate, grown in multilineage culture conditions. Gain- and loss-of-function experiments revealed that hsa-miR-299-5p participates in the regulation of hematopoietic progenitor fate, modulating megakaryocytic-granulocytic versus erythroid-monocytic differentiation

2010 - Integrative genomics analyses reveal molecularly distinct subgroups of B-cell chronic lymphocytic leukemia patients with 13q14 deletion [Articolo su rivista]
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

PURPOSE: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.EXPERIMENTAL DESIGN: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.RESULTS: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.CONCLUSIONS:Overall, our data highlight the presence of 2 distinct molecular types of 13q14 deletions, which may be of clinical relevance in CLL

2010 - Metabotropic glutamate receptor-4 modulates adaptive immunity and restrains neuroinflammation. [Articolo su rivista]
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

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

2010 - PTPN11 mutations in childhood acute lymphoblastic leukemia occur as a secondary event associated with high hyperdiploidy [Articolo su rivista]
Molteni CG; Te Kronnie G; Bicciato S; Villa T; Tartaglia M; Basso G; Biondi A; Cazzaniga G

The protein tyrosine phosphatase, non-receptor type 11 (PTPN11) gene encodes SHP-2, a phosphatase involved in many signaling pathways, with a key role in development and hematopoiesis. Somatic PTPN11 mutations were found with variable prevalences in pediatric leukemia, most frequently associated with juvenile myelomonocytic leukemia (JMML).1,2 In vitro and in vivo studies, mainly in a myeloid context, demonstrated a crucial involve- ment of PTPN11 mutations in the hyperactivation of the RAS/ERK pathway.3 The oligoclonality of transgene integration sites, and the long latency before overt leukemia in mice transduced with mutant PTPN11,3 suggested that additional molecular lesions are needed to cooperate with PTPN11 mutations in leukemogenesis. This observation is in line with the ‘two hit model’ proposed by Greaves4 for B-cell precursor childhood acute lymphoblastic leukemia (ALL), in which an initiating alteration, often occurring prenatally, gives rise to a preleukemic clone, which eventually becomes fully malignant by subsequent acquisition of other molecular alterations.

2010 - Prognosis of breast cancer patients by monitoring the expression of two genes [Brevetto]
S. Piccolo; M. Cordenonsi; M. Adorno; S. Bicciato

The invention is related to a method for evaluating a breast cancer patient’s risk of recurrence comprising detecting the level of CyclinG2 (Gene ID = 901) gene expression alone or in combination with Sharp1 (Gene ID = 79365) in a sample.The detection comprises measuring a signal directly related to the gene(s) expression in said sample, acquiring the signal and evaluating the risk of cancer recurrence of a breast cancer patient by: -calculating a signature score for Cyclin G2 gene expression values alone or for, preferably, both Cyclin G2 and Sharp-1 expression values in the unknown sample, wherein said signature score is defined as: being K=1 when using Cyclin G2 alone and K=2 when using both Cyclin G2 and Sharp-1, the expression level of CyclinG2 or Sharp-1 in the unknown sample i, and respectively the estimated mean and standard deviation values of the Cyclin G2 and/or Sharp-1 expression levels in a population with known clinical history, and wherein a signature score lower than zero indicates an increased risk of breast cancer recurrence.The detection may be carried out by molecular and/or immunological means, where by molecular means are meant assays based on nucleic acids such as PCR, microarray analysis or Northern-blot.The method further comprises statistical analysis of the signal through the following steps:-quality control of the acquired signal, -signal normalization; -optionally rescaling the acquired signal, and is preferably carried out by a software run on a computer.The invention further provides for a kit to evaluate Cyclin G2 expression alone or in combination with Sharp 1 and determine the risk of cancer recurrence in a sample from a breast cancer patient, said kit preferably comprising:-a CyclinG2- specific reagent, preferably a oligonucleotide consisting in a oligonucleotide comprising at least a 13-mer oligonucleotide derived from SEQIDNO:1 or its complementary sequence; -a Sharp1-specific reagent, preferably a oligonucleotide consisting in a oligonucleotide comprising at least a 13-mer oligonucleotide derived from SEQIDNO:2 or its complementary sequence;-instruction for calculating the signature score of the unknown sample and classifying the unknown sample in the minimal signature Low group when its signature score is negative or in the minimal signature High when its signature score is positive, according to calculation defined for the method above, wherein classification into the minimal signature Low group is an indication of an high risk of cancer recurrence for a breast cancer patient.According to a preferred embodiment said instruction are carried out by a software.Optionally the kit may further comprise as reference standards, CyclinG2 and Sharp1 standard expression controls High and Low, as expression values or as nucleic acid samples. Said expression values or nucleic acid samples are preferably derived respectively from a non metastatic breast cancer cell line and/or from a highly metastatic cell line.

2010 - Tumor-induced tolerance and immune suppression depend on the C/EBPbeta transcription factor [Articolo su rivista]
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 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

2009 - A Mutant-p53/Smad Complex Opposes p63 to Empower TGFβ-Induced Metastasis [Articolo su rivista]
M. Adorno; M. Cordenonsi; M. Montagner; S. Dupont; C. Wong; B. Hann; A. Solari; S. Bobisse; M.B. Rondina; E. Guzzardo; A.R. Parenti; A. Rosato; S. Bicciato; A. Balmain; S. Piccolo

TGFβ ligands act as tumor suppressors in early stage tumors but are paradoxically divertedinto potent prometastatic factors in advanced cancers. The molecular nature of this switchremains enigmatic. Here we show the workings of a previously undescribed pathway in whichTGFβ-dependent cell migration, invasion and metastasis are empowered by mutant-p53 andopposed by p63. Mechanistically, TGFβ acts in concert with oncogenic Ras and mutant-p53 toinduce the assembly of a mutant-p53/p63 protein complex in which Smads serve as essentialplatforms. Within this ternary complex, p63 functions are antagonized. Downstream of p63,we identified two novel metastasis suppressor genes associated with metastasis risk in a largecohort 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-upwith particular metastasis proclivity by TGFβ-dependent inhibition of p63 function.

2009 - A computational procedure to identify significant overlap of differentially expressed and genomic imbalanced regions in cancer datasets. [Articolo su rivista]
Bicciato S; Spinelli R; Zampieri M; Mangano E; Ferrari F; Beltrame L; Cifola I; Peano C; Solari A; Battaglia C.

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

2009 - A-MADMAN: annotation-based microarray data meta-analysis tool. [Articolo su rivista]
Bisognin A; Coppe A; Ferrari F; Risso D; Romualdi C; Bicciato S; Bortoluzzi S

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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

2009 - Identification of microRNA expression patterns and definition of a microRNA/mRNA regulatory network in distinct molecular groups of multiple myeloma [Articolo su rivista]
Lionetti M; Biasiolo M; Agnelli L; Todoerti K; Mosca L; Fabris S; Sales G; Deliliers GL; Bicciato S; Lombardi L; Bortoluzzi S; Neri A

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.

2009 - Integration of Genomic and Gene Expression Data of Childhood ALL Without Known Aberrations Identifies Subgroups with Specific Genetic Hallmarks [Articolo su rivista]
S. Bungaro; M.C. Dell'Orto; A. Zangrando; D. Basso; T. Gorletta; L. Lo Nigro; A. Leszi; B.D. Young; G. Basso; S. Bicciato; A. Biondi; G.T. Kronnie; G. Cazzaniga

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 nuclecitide 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)/12p 13 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. SMAD 1 emerged as a down-regulated gene in CDKN2A homozygous deleted cases compared with nondeleted. The JAG1 gene, encoding the jagged I 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.

2009 - Methods for interpreting lists of affected genes obtained in a DNA microarray experiment [Articolo su rivista]
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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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

2009 - Microarray data mining using Bioconductor packages [Articolo su rivista]
Nie H; Neerincx PB; van der Poel J; Ferrari F; Bicciato S; Leunissen JA; Groenen MA

BACKGROUND: 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. RESULTS: 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). CONCLUSION: 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.

2009 - Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production. [Articolo su rivista]
Carata E; Peano C; Tredici SM; Ferrari F; Talà A; Corti G; Bicciato S; De Bellis G; Alifano P.

Background: 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.Results: 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.Conclusion: 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.

2008 - Approccio integrato di biologia sistemica per la ricostruzione dei processi di segnalazione molecolari e di attivazione monocitaria/macrofagica in risposta a stimoli infiammatori in condizioni fisiologiche mediante tecnologie omiche [Partecipazione a progetti di ricerca]
C. Battaglia; D. Cavalieri; S. Bicciato; A. Grande; P. Mauri

A) La definizione di un modello d'attivazione in vitro di monociti/macrofagi rappresentativo dell'infiammazione in condizioni normali;B) Ottenimento di un modello cellulare trasferibile per lo studio del comportamento cellulare di monociti d'individui affetti da malattie infiammatorie croniche oautoimmuni;C) l'identificazione di una mappa trascrittomica costituita dai geni, microRNA espressi in modo differenziale nel modello d'attivazione dei monociti;D) L'identificazione della mappa del proteoma/secretoma derivante dai campioni biologici in studioE) La caratterizzazione delle funzioni di MafB nella biologia dei monociti / macrofagi e dei suoi bersagli molecolariF) L'identificazione di siti putativi di legame per MafB sui promotori di geni correlati al differenziamento e all'attivazione monocito / macrofagicaG) La produzione di una banca dati integrata con dati genomici e proteomici relativi all'attivazione monocitica/macrofagica;H) L'identificazione di regioni genomiche differenzialmente espresse o arricchite in specifiche caratteristiche;I) La ricostruzione delle reti d'interazione molecolare nell'attivazione monocitica/macrofagicaJ) Fornire una cornice computazionale per l'integrazione di dati prodotti in questo studio con informazioni biologiche esistenti con un approccio integrato genomico,proteomico e bioinformatico per decifrare il complesso sistema biologico alla base dei processi d'infiammazione e della risposta immunitaria innata;K) Lo sviluppo di un'approccio metodologico per decifrare un sistema biologico complesso come quello della risposta immunitaria innata che possa esseretrasferibile a un modello di studio di patologie infiammatorie croniche e delle malattie autoimmuni, con l'obiettivo di trovare nuovi farmaci.

2008 - Development of a bioinformatics framework for the analysis of complex biological systems: application to the study of myeloid differentiation [Partecipazione a progetti di ricerca]
S. Bicciato; S. Bortoluzzi; S. Ferrari; F. Ferrari; E. Tagliafico; D. Lancet; M. Safran

The project aims at establishing the methodological bases and developing the computational infrastructures to investigate, through a systems biology approach, the regulatory network characterizing myeloid cell differentiation.The methodological aims include the development of innovative computational methods to study gene expression regulation at transcriptional and post-transcriptional levels.The applicative aim regards the study of genetic and epigenetic mechanisms of regulation of genome expression in myeloid cells, to reconstruct regulatory networks involved in differentiation.Strategically, the project aims at starting-up a bioinformatics core unit which, in collaboration with a top-ranking international research institution, will support and complement the preexisting biomedical expertise at the University of Modena, facilitating high level post-genomic research and broadening the funding opportunities.

M. Lionetti; L. Agnelli; L. Mosca; D. Ronchetti; S. Fabris; A. Andronache; K. Todoerti; D. Verdelli; P. Ponzo; S. Bicciato; G. Lambertenghi-Deliliers; A. Neri

The role of microRNAs (miRNAs) in multiple myeloma (MM) remainsto be fully elucidated. To identify miRNAs potentially deregulated inMM, we performed an integrative analysis of genome-wide DNA copynumber (CN), gene expression profiling and miRNAs expression profilingin a panel of 16 human myeloma cell lines (HMCLs). Global miRNAand mRNA expression data were generated on Agilent miRNA microarrays(representing 470 human mature miRNAs) and AffymetrixGeneChip HG-U133A arrays, respectively; genome-wide profiling datawere generated on Affymetrix GeneChip Human Mapping 250K Nsparrays. CN values were inferred using the DNAcopy R Bioconductorpackage. To measure the correlation between the expression levels ofeach miRNA and the corresponding CN values, non-parametric analyseswere performed (Kendall's tau correlation). We identified 22 miRNAswith a good correlation (p<5×10–2) between expression levels and localCN variations, suggesting that a dosage effect could be a main mechanismunderlying their expression. The identified miRNAs are mappedwithin different genomic regions, including chromosome 22q11.21,8q24.22, 17, 7 and 16q22, and for some of them a role in other types ofcancer as well as in MM has already been reported or suggested. Thesame approach was applied in order to investigate the associationbetween intronic miRNA and corresponding host-gene expressions. Apositive correlation (p<5×10–2) was found for 22 intronic miRNAs mappedwithin 20 different deregulated host genes, strongly supporting the suggestionthat intronic miRNAs and their host genes share the same regulatorysequences and can be co-transcribed. Among the most correlatedmiRNA/host-gene pairs we identified miR-342-3p/EVL and miR-335/MEST, for which the miRNA expression levels were validated bymeans of Q-RT-PCR. The presence of a correlation was further confirmedin a fraction of primary tumors. Some miRNA/host-genes expressionalso shared a significant correlation with CN variations. In conclusion, wedemonstrated that the presence of genomic lesions and the expression ofhost-genes can affect miRNA expression in HMCLs. We are nowattempting to investigate these findings in primary tumors in order to betterdefine the role of miRNAs in the pathogenesis of multiple myeloma.

L. Agnelli; S. Fabris; S. Bicciato; D. Lambertenghi; A. Neri

marrowplasma cells that accounts for 10% of all hematological malignancies.The broad clinical spectrum of plasma cell dyscrasias range from apre-malignant condition termed monoclonal gammopathy of undeterminedsignificance (MGUS) to smouldering MM (SMM), truly overt andsymptomatic MM, and extra-medullary myeloma/plasma cell leukemia(PCL).1,2 MM is characterised by a profound genomic instability thatinvolves both ploidy and structural rearrangements.3 Nearly half of MMtumours are hyperdiploid (H-MM) characterised by recurrent trisomies,particularly of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21; the non-hyperdiploid(NH-MM) tumours are cases with a hypodiploid, pseudodiploidor near tetraploid chromosome number, and are frequently associatedwith chromosome 13 deletions and immunoglobulin heavy chain (IGH)locus translocations involving a promiscuous number of partner loci,mainly CCND1 (11q13), FGFR3/MMSET(4p16.3), MAF (16q24), MAFB(20q) or CCND3 (6q).4-7 It has also been demonstrated that almost allMM patients are affected by deregulation of one of the cyclin D genes(CCND1, 2 or 3), which may therefore play an important role in themolecular pathogenesis of the disease.MM patients can be stratified into five molecular groups on the basisof the presence of known IGH translocations and cyclin D deregulation(TC classification): TC1 is characterised by t(11;14) or t(6;14); TC2 isassociated with hyperdiploidy and low-moderate levels of CCND1 inthe absence of IGH translocations; TC3 includes tumours not fallinginto any of the other groups, most of which express CCND2; TC4 isassociated with t(4;14) and high CCND2 levels; and TC5 expresses thehighest levels of CCND2 in association with either t(14;16) or t(14;20).8,9We have combined fluorescence in situ hybridization (FISH) analysesand global gene expression profiling (GEP) in a series of studies aimedat elucidating the transcriptional profiles associated with plasma celldyscrasias in a panel of newly diagnosed patients including 11 withMGUS, 132 with MM and nine with PCL. The unsupervised analysis ofthe gene expression data profiled on high-density oligonucleotidemicroarrays identified two major groups: one including the majority ofMGUS patients and normal controls, and the other all the PCL and mostof the MM cases. Therefore, neither the MGUS, nor the PCL and MMsamples could be identified as distinct entities. A multi-class analysisrevealed probe sets specifically distinguishing MGUS from PCL, with theMM cases showing their progressively modulated expression. TheMGUS cases showed the up-regulation of immune response genes,whereas the PCL cases showed the positive modulation of primarymetabolism, and cell cycle and apoptosis induction. The hierarchicalclustering generated in the 132 MM database was mainly driven bygroups reflecting the TC classification.We also analysed the GEP data in the context of distinct genetic lesionsinvolving chromosomal gains or losses. All of the del(13) cases showed67 down-regulated genes involved in protein biosynthesis, ubiquitinationor transcriptional regulation, most of which (44/67) mapped alongthe whole chromosome 13.10 In terms of 1q gain, the differential expressionof 61 genes mainly localised on chromosome 1q12-1q44 distinguishedMM patients with or without 1q extra copies. Functional analysisof the identified genes revealed their involvement in energy productionpathways, intracellular protein transport, and stress-induced endoplasmicreticulum responses.11Finally, the differential expression of 225 genes mainly involved inprotein biosynthesis, transcriptional machinery and oxidative phosphorylationdistinguished H-MM from NH-MM. Most of the up-regulatedgenes in H-MM mapped to the chromosomes involved in hyperdiploidy,whereas a significant fraction of the genes in NH-MM mapped to 16q.12Overall, the GEP data suggested a widespread gene-dose effect as theimbalances in expression closely correlated with the genomic structuralabnormalities.These results prompted

2008 - Unexpected structural and functional consequences of the R33Q homozygous mutation in cardiac calsequestrin - A complex arrhythmogenic cascade in a knock in mouse model [Articolo su rivista]
N. Rizzi; N. Liu; C. Napolitano; A. Nori; F. Turcato; B. Colombi; S. Bicciato; D. Arcelli; A. Spedito; M. Scelsi; L. Villani; G. Esposito; S. Boncompagni; F. Protasi; P. Volpe; S.G. Priori

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

2007 - Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays [Articolo su rivista]

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.

2007 - Gene expression profile of cerebrospinal fluid mononuclear cells in oligoclonal band positive and oligoclonal band negative multiple sclerosis patients at early stage of disease [Abstract in Rivista]

Different blood and CSF T and B lymphocyte subsets are thought to be involved in multiple sclerosis (MS) pathogenesis. Microarray technology provides a tool for large-scale analysis of gene expression programs. Microarray analysis of CSF inflammatory cells may improve our understanding of the unique immunological context within the CNS compartment.

2007 - Genome-wide analysis of DNA copy number changes in multiple myeloma using high-density SNP arrays [Abstract in Rivista]
Mosca L; Agnelli L; Fabris S; Ronchetti D; Todoerti K;Lionetti M; Kwee I; Bicciato S; Baldini L; Morabito F; Bertoni F;Lambertenghi Deliliers G; Neri A

Introduction. Multiple myeloma (MM) is characterized by deep genomicinstability that involves both ploidy and structural rearrangements.Nearly half of MM tumors are non-hyperdiploid and frequently showchromosome 13 deletion and the common chromosomal translocationsinvolving the immunoglobulin heavy chain (IGH) locus on chromosome14q32. The remaining tumors are hyperdiploid, showing low prevalenceof IGH translocations and chromosome 13 deletions. Despite therecent advances, the spectrum of genetic lesions leading to biological andclinical variability of MM has not been defined yet. Our study wasaimed at defining a genome-wide pattern of genetic lesions in a representativeand stratified panel of MM patients using novel high-throughputapproaches, to provide insights into the genomic heterogeneity associatedwith plasma cell neoplasms. Methods. Genome-wide profilingdata of 57 MM patients, 7 plasma cell leukemia (PCL) and 14 normalsamples were generated on the GeneChip® Human Mapping 50K XbaSNP arrays. The local DNA copy number variations were calculatedusing the DNAcopy Bioconductor package. The gene expression profilesof the tumor samples were generated on the GeneChip® HG-U133Aarrays. Hierarchical clustering algorithms were applied to identify thenatural grouping of gene expression and genome profiles. Results. Anunsupervised analysis on the genome-wide profiles of our dataset showed that chromosome 1q gains, chromosome 13 deletions andhyperdiploidy are the main genetic aberrations driving samples grouping.High frequencies of increased DNA copy number levels were foundin six extended regions, specifically chromosome 1q and those chromosomesinvolved in hyperdiploidy (7, 9, 11, 15 and 19). As regards theregions with decreased copy number, we found the involvement of thechromosomal regions 6q and 16q, together with the newly reported 18qand 4p that warrant further investigations. The depicted clusteringstrongly correlated with that generated on the gene expression profilesof the patients in the dataset. Furthermore, we identified different geneswith altered expression level corresponding to local copy number variations.Discussion. Our results showed that genomic structural abnormalitiesin multiple myeloma closely reflected in expression imbalances.Our data reinforce the importance of using novel high-throughputapproaches to provide insights into the characterization of novel potentialgenetic lesion in primary myeloma tumors.

2007 - Genomic expression during human myelopoiesis [Articolo su rivista]
Ferrari F; Bortoluzzi S; Coppe A; Basso D; Bicciato S; Zini R; Gemelli C; Danieli GA; S Ferrari

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

2007 - Genomica e proteomica computazionale [Curatela]
R. Bellazzi; S. Bicciato; S. Cavalcanti; C. Cobelli; G.M. Toffolo

Le sequenze geniche, il modo in cui esse si esprimono e vengono tradotte in proteine, la natura di tali proteine e la loro funzione sono alla base della fisiologia della cellula e determinano il comportamento a livello di organo e di organismo in risposta ad uno stimolo esterno o durante una malattia. Nell’ultimo decennio lo sviluppo di nuove tecnologie di biologia molecolare per lo studio del genoma e del proteoma ha aperto all’industria della salute degli orizzonti prima inimmaginabili, che tuttavia necessitano dello sviluppo di metodologie adeguate all’analisi dei dati. In particolare, le tecnologie per il sequenziamento e il monitoraggio del trascrittoma e del proteoma generano un’enorme quantità di dati che è necessario gestire utilizzando le tecnologie informatiche adeguate, analizzare e interpretare in maniera corretta dal punto di vista statistico/matematico e coerente con il reale significato biologico dei dati stessi. È inoltre indispensabile confrontare ed integrare l’informazione ottenuta da questi dati con l’informazione biologica nota, reperibile in maniera non sempre ordinata ed automatica nelle decine di data base biologici presenti in rete. Dopo un’introduzione sulla biologia molecolare e sull’impatto delle nuove tecnologie high-throughput in medicina e biologia, il volume tratta le metodologie per l’analisi delle sequenze geniche, del trascrittoma e del proteoma e la loro integrazione in approcci “systems biology”. L’impiego delle metodologie e tecnologie e’ illustrato in due settori applicativi di grande importanza, l’oncogenomica e la farmacogenetica/genomica. Infine vengono discussi aspetti relativi all’integrazione da data-base biologici e l’impiego delle architetture informatiche nel processo di analisi dei dati.Introduzione - tutorial: geni, proteine e cellule - biologia e medicina molecolare - tecnologie high throughput - metodi di analisi di sequenze e genoma - modelli e metodi per l’analisi del trascrittoma - modelli e metodi per l’analisi del proteoma - systems biology - oncogenomica e farmacogenomica - tecnologie bioinformatiche.

2007 - Integrative genomic analysis reveals distinct transcriptional and genetic features associated with chromosome 13 deletion in multiple myeloma [Articolo su rivista]

Background and Objectives The chromosome 13 deletion (Delta(13)) is one of the most frequent chromosomal alterations in multiple myeloma (MM). Delta(13) 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 Delta(13) 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.Results 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 Delta(13): 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 Delta(13)-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 Delta(13)-positivity and the presence of extra copies of 1q21-1q42 (p=6x10(-4)) or the absence of chromosome 11 and 19 trisomy (p=5x10(-4)).Interpretation and Conclusions Our results indicate that distinct types of chromosomal aberrations are closely related to the transcriptional profiles of Delta(13)-positive cases, suggesting that the contribution of Delta(13) to the malignancy should be considered together with associated abnormalities.

2007 - Molecular characterization of human multiple myeloma cell lines by integrative genomics: Insights into the biology of the disease [Articolo su rivista]

TTo 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 WHSCI/MMSET gene involved in t(4; 14)(p 16;q32) was expressed at different levels in all of the HMCL; 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 I q in 88% of the analyzed cell lines, together with recurrent gains on 8q, 18q, 7q, and 20q; the most frequent deletions affected I p, 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.

2007 - Novel definition files for human GeneChips based on GeneAnnot [Articolo su rivista]

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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, along with supplementary information (CDF libraries, installation guidelines and R code, CDF statistics, and analysis results).

2007 - Transcriptional features of multiple myeloma patients with chromosome 1q gain [Articolo su rivista]

Abnormalities of chromosome 1 are among the mostfrequent chromosomal alterations in multiple myeloma(MM), being found in up to 45% of patients.1,2 It has beenreported that the short arm of chromosome 1 is preferentiallyinvolved in deletions, whereas the long arm is associatedwith amplification. The gain of 1q (1q/gain) can occur asisochromosomes, duplications or jumping translocations. Ithas been widely reported that 1q/gain MM patients arecharacterized by complex karyotypes and aggressive disease,and a close association with poor-risk genetic features, suchas chromosome 13q deletion (D13) and the t(4;14) translocationhas also been described.1 It has been recentlydemonstrated that gains/amplification of 1q21 increase asthe condition goes from smoldering to overt MM, thussuggesting that these regions contain critical genes for diseaseprogression.2 These findings along with the limited informationconcerning specific transcriptional profiles prompted us tomolecularly characterize 1q/gain MMs by FISH and microarrayanalyses.

2007 - Transcriptional features of multiple myeloma patients with chromosome IQ gain [Abstract in Rivista]
Fabris S; Ronchetti D; Agnelli L; Baldini L; Morabito F; Bicciato S; Basso D; Todoerti K; Lombardi L; Lambertenghi-Deliliers G; Neri A

Introduction. Abnormalities of chromosome 1 are among the most frequentchromosomal alterations in MM (45% of patients). The long armof chromosome 1 is associated with amplification (1q/gain) that canoccur as isochromosomes, duplications or jumping translocations.1q/gain MM patients are characterized by complex karyotypes andaggressive disease, and their number increases as the condition goesfrom smoldering to overt MM, suggesting that these regions containcritical genes for disease progression. These findings along with the limitedinformation concerning specific transcriptional profiles, promptedus to molecularly characterize 1q/gain MMs by FISH and microarrayanalyses. Methods. Purified plasma cells from 77 MMs at diagnosis werecharacterized by FISH for the presence of 11 polisomy, the most recurrentIGH translocations, ploidy status, chromosome 13 deletion, and byglobal gene expression profiling using the Affymetrix U133A arrays.Assessment of 1q/gain by FISH was performed by using three BACclones specific for the BCL9 (1q21.1), CKS1B (1q22) and ARF1 (1q42.13)loci, and setting the threshold as 10%. Results. 1q/gain was identified in40/77 (51.9%) patients; three (75%) or four (12.5%) signals of all the 1qprobes were found in 35 patients and, in the remaining five samples(12.5%), the probes mapping to 1q21 and 1q22 showed more signalsthan that mapping to 1q42. 1q/gain was observed in the majority ofpurified plasma cells (median 96%) in all but three patients (range 12-20%). 1q extra copies significantly correlated with chromosome 13 deletion(p<10-4), and the absence of chromosome 11 polisomy (p=0.038),but not with ploidy status (p=0.0971). The differential expression of 61genes (mainly localized on chromosome 1q12-1q44) distinguishes MMpatients with or without 1q/gain. Functional analysis of the identifiedgenes revealed their involvement in energy production pathways, intracellularprotein transport, and endoplasmic reticulum-stress inducedresponses. The transcriptional fingerprint was robustly validated on apublicly available gene expression dataset, with a global classificationrate of 85.2% for the independent cohort of MM cases. Discussion. Thesedata improve our knowledge concerning the specific genes/pathwaysderegulated by 1q abnormalities, and provide a promising focus for furtherstudies aimed at defining new therapeutic strategies in MM.

2007 - Transcriptional profiles in melanocytes from clinically unaffected skin distinguish the neoplastic growth pattern in patients with melanoma [Articolo su rivista]
C. Magnoni; E. Tenedini; F. Ferrari; L. Benassi; C. Bernardi; G. Gualdi; G. Bertazzoni; E. Roncaglia; L. Fantoni; R. Manfredini; S. Bicciato; S. Ferrari; A. Giannetti; E. Tagliafico

Background It is generally accepted that sunlight may contribute to the development of melanoma. Objectives To analyse gene expression of melanocytes obtained from clinically unaffected skin of patients with melanoma and healthy controls before and after exposure to ultraviolet B radiation. Methods Using GeneChip array technology, the gene expression of melanocytes obtained from the two donor groups was profiled, in order to identify transcriptional differences affecting susceptibility to melanoma. Results The data collected did not show any difference between the expression profiles of melanocytes purified from normal donors and from patients with melanoma that was able to give a statistically significant class separation. However, by means of unsupervised clustering our data could be divided into two main classes. The first class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a vertical growth phase (VGP) melanoma, while the second class included the transcriptome profiles of melanocytes obtained from skin samples of patients with a radial growth phase (RGP) melanoma. Conclusions These data suggest that melanocytes in patients with VGP and RGP melanomas show significant differences in gene expression profiles, which allow us to classify patients with melanoma also from clinically unaffected skin.

2007 - Upregulation of translational machinery and distinct genetic subgroups characterise hyperdiploidy in multiple myeloma [Articolo su rivista]

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.

2006 - A gene expression signature associated with survival in metastatic melanoma [Articolo su rivista]

Background: 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.Methods: 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.Results: 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.Conclusion: 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.

2006 - A locally adaptive statistical procedure (LAP) to identify differentially expressed chromosomal regions [Articolo su rivista]

Motivation: The systematic integration of expression profiles and other types of gene information, such as chromosomal localization, ontological annotations and sequence characteristics, still represents a challenge in the gene expression arena. In particular, the analysis of transcriptional data in context of the physical location of genes in a genome appears promising in detecting chromosomal regions with transcriptional imbalances often characterizing cancer.Results: A computational tool named locally adaptive statistical procedure (LAP), which incorporates transcriptional data and structural information for the identification of differentially expressed chromosomal regions, is described. LAP accounts for variations in the distance between genes and in gene density by smoothing standard statistics on gene position before testing the significance of their differential levels of gene expression. The procedure smoothes parameters and computes p-values locally to account for the complex structure of the genome and to more precisely estimate the differential expression of chromosomal regions. The application of LAP to three independent sets of raw expression data allowed identifying differentially expressed regions that are directly involved in known chromosomal aberrations characteristic of tumors.

2006 - Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data [Articolo su rivista]

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.

2006 - From medical to biological informatics: Searching for diagnostic markers in Parkinson's disease patients' lymphocytes using transcriptomics [Abstract in Rivista]

Objective: To determine whether the transcriptomic profile of PD patients’s lymphocytescould be used in separating them from the healthy (with respect to PD) subjects.Background: To-date, the diagnosis of Parkinson’s disease (PD) is based primarily onclinical symptoms. These symptoms, however, are not specific for the disease, since theycharacterize the clinical syndrome of parkinsonism in general, the latter being originated fromother disorders as well. It becomes apparent then that there is a need for accurate, diseasespecific,non- or least-invasive diagnostic tools for PD. Peripheral blood lymphocytes (PBLs)are a readily accessible peripheral tissue. They have been studied in the context of brainresearch, because it is believed that they could provide leads into the biochemistry,physiology and pathophysiology of the central nervous system (CNS), as they exhibitbiochemical similarities with their CNS counterparts.Methods: We analyzed the blood samples of a group of 7 idiopathic first-time diagnosedPD patients prior to the initiation of any treatment, at the transcriptional level using fullgenomeDNA microarrays of Affymetrix platform. The acquired data were subsequentlyinvestigated using multivariate statistical analysis techniques.Results: Combination of the microarray analysis results with any available informationfrom the medical history of the patients and their diagnosis based on the clinical symptomsrevealed the importance of the integration of medical with the biological informatics to (a)correctly select the control subjects of the study, and, especially in the case of diagnostic toolsbased on blood samples, (b) isolate the disease-specific markers/molecular differences fromany other medical history that could contribute to the particular transcriptomic profile.Conclusions: Analyzing high-throughput –omic data in the context of the patients’medical history is indeed very significant towards the goal of the modern biomedicine forpersonalized diagnosis and therapy.

Tenedini, Elena; Tagliafico, Enrico; Manfredini, Rossella; Ferrari, Francesco; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Tatiana Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Ferrari, Sergio

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to All-Trans Retinoic-Acid (ATRA) and Vitamin-D3 (VD) and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature of 11 genes, among them is particularly intriguing the presence of Meis1 and ID3, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients known to have poor prognostic significance.

2006 - Identification of a molecular signature predictive of sensitivity to differentiation induction in acute myeloid leukemia [Articolo su rivista]
Tagliafico, E.; Tenedini, E.; Manfredini, R.; Grande, A.; Ferrari, F.; Roncaglia, E.; Bicciato, S.; Zini, R.; Salati, S.; Bianchi, E.; Gemelli, C.; Montanari, M.; Vignudelli, T.; Zanocco-Marani, T.; Parenti, S.; Paolucci, P.; Martinelli, G.; Piccaluga, P.P.; Baccarani, M.; Specchia, G.; Torelli, U.; Ferrari, S.

Acute myeloid leukemia (AML) blasts are immature committed myeloid cells unable to spontaneously undergo terminal maturation, and characterized by heterogeneous sensitivity to natural differentiation inducers. Here, we show a molecular signature predicting the resistance or sensitivity of six myeloid cell lines to differentiation induced in vitro with retinoic acid or vitamin D. The identified signature was further validated by TaqMan assay for the prediction of response to an in vitro differentiation assay performed on 28 freshly isolated AML blast populations. The TaqMan assay successfully predicts the in vitro resistance or responsiveness of AML blasts to differentiation inducers. Furthermore, performing a meta-analysis of publicly available microarray data sets, we also show the accuracy of our prediction on known phenotypes and suggest that our signature could become useful for the identification of patients eligible for new therapeutic strategies.

2006 - Identification of new genomic lesions in childhood ALL without known genetic aberrations: A microarray study of gene expression and genotype data. [Abstract in Rivista]

Despite the risk stratification based on the prognostic relevant translocations, approximately 20-25% of childhood ALL patients cannot be classified according to genetic hallmarks. Recent studies based on single nucleotide polymorphism (SNP) analyses showed that Loss of Heterozygosity (LOH), with or without loss of genetic material, is frequently related to childhood ALL and AML. Moreover, there is an increasing interest in integrating the gene expression profiles with chromosomal localizations to identify new genetic subgroups. We applied an integrated approach composed of DNA index estimation, PCR (and/or RT-PCR) and cytogenetics, to exclude patients with known molecular and cytogenetics aberrations. Aim of the study was to identify criptic abnormalities in childhood ALL patients by performing an integrative analysis of gene expression and copy number changes (CNC) data. The 30 patients included in our study met the following inclusion criteria: a) B-cell precursor childhood ALL b) DNA index 1; c) negativity at t(4;11), t(12;21), t(9;22), t(1;19) RT-PCR screening; d) cytogenetics revealing normal karyotype or not technically feasible. Patients genomic DNA has been analyzed by the Affymetrix GeneChip Mapping 100K SNP arrays to identify genomic regions of LOH and CNC. In parallel, biotin-labeled cRNA has been synthesized and hybridized on HG-U133Plus2 Probe Arrays in accordance with the MILE (mircroarray innovation in leukemia) protocols; all samples were part of the B-ALL class without known genetic aberrations.The presence of del(9)(p21) was found in 9/30 (30%) patients, with an homozygous commonly deleted region involving CDKN2A and CDKN2B genes in 7/30. Hemizygous losses on 9p13 were found in two cases. Four patients (13,3%) showed the hemizygous deletion of chromosome 12p13.2, with a commonly deleted region involving the ETV6 gene. In one ETV6 deleted patient FISH analysis identified a translocation involving the first ETV6 exon and an unknown partner gene. Four patients, including three ETV6 deleted, showed the presence of the intrachromosomal amplification of chromosome 21 (iAMP21) with a common region of amplification between 31.3-43.5 Mb and a common region of deletion in 2/4 patients between 43.5-47 Mb. Four patients showed large regions of LOH not associated with CNC. Three patients did not show CNC and random microdeletions were found in single cases. Remission samples were analyzed for 5 patients carrying different aberrations, showing small regions of LOH in both the diagnosis and remission samples in 4 cases. No CNC was found in the remission samples. We found a statistically significant reduction for the expression of the CDKN2A transcript located within the (9)(p21) region. We are applying a locally adaptive statistical procedure to identify genes whose expression signals are specifically modulated by CNC. The integration of genomic variation and gene expression profiling might may be useful to identify new hidden genetic lesions, and to learn how critical regulators of tumor are linked to genomic alterations in cancer cells.

2006 - Identification of specific transcriptional patterns associated with hyperdiploidy in multiple myeloma. [Abstract in Rivista]

Karyotypic instability is strongly associated with multiple myeloma (MM). According to the chromosome number pattern, two major groups are recognized: hyperdiploid (H) tumors, associated with recurrent trisomies involving non-random chromosomes (3, 5, 7, 9, 11, 15, 19 and 21); and non hyperdiploid (NH) tumors associated with hypodiploid, pseudodiploid or near-tetraploid karyotypes. MM patients are approximately equally distributed between the two categories; notably, the most recurrent IGH translocations and chromosome 13 deletion appear to be prevalently associated with NH-MM, whereas recent evidences have suggested that H-MM correlates with a favorable prognosis. To molecularly characterize these two genetic categories, we performed a gene expression profiling analysis on 66 newly-diagnosed MM, characterized by FISH analyses for IGH translocations, 13q14 deletions and additional copies of chromosomes 1, 11 and 19. The ploidy status was investigated by combining two recently proposed FISH approaches (Wuilleme S. et al., 2004; Chng W.J. et al., 2005). The gene expression profiles of highly purified MM plasma cells have been generated by means of high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed using unsupervised and supervised approaches (two-dimensional hierarchical clustering and SAM, respectively). The differential expression of 229 genes distinguished the 28 H-MM from the 38 NH-MM cases. The 208 upregulated genes in H-MM mapped mainly on the chromosomes involved in hyperdiploidy, while a significant percentage (29%) of the 21 genes upregulated in NH-MM were localized on 16q. The identified transcripts have been further validated on a publicly available gene expression dataset of an independent cohort of 64 MM patients (Carrasco et al., 2005). Notably, the global classification rate for the 64 cases resulted of 81%, confirming the validity of the identified transcriptional fingerprint. A functional analysis revealed a significant fraction of genes involved in protein biosynthesis (38%), transcriptional machinery and oxidative phosphorylation. Furthermore, an integrative genomic approach using a model-free statistical method (LAP, locally adaptive statistical procedure) supported these findings, allowing the identification in H-MM of globally upregulated regions on the chromosomes 3, 5, 9, 15 and 19, along with the downregulation of a region on 16q arm. Remarkably, two sub-groups are clearly distinguishable within H-MM group, one associated with chromosome 11 gain and the other showing 1q gain and chromosome 13 deletion. A supervised analysis of the H-11 vs H-13/1 patients identified 57 differentially expressed genes. Eleven of the 18 genes up-regulated in the H-11 group mapped to chromosome 11, whereas 21 of the 39 genes up-regulated in the H-13/1 group mapped to the 1q region. Notably, CCND2 resulted the most significantly upregulated gene in H-13/1 group. Our data reinforce the importance of combining cytogenetics and gene expression approaches for a better definition of the genetic alterations in MM and provide a molecular and genomic framework for dissection of disease pathogenesis and clinical management.

2006 - Immunophenotype signature as a tool to define prognostic subgroups in childhood acute myeloid leukemia [Articolo su rivista]

Acute myeloid leukemia (AML) is a heterogeneous diseasegroup morphologically classified, based on the French–American–British (FAB) classification, into eight main subgroupsdefined as subtypes M0–M7. Besides morphologic differences,genetic abnormalities have been recognized; cytogenetics andmolecular analyses are currently used to identify subgroups ofAML with different clinical prognosis. However, in spite ofavailable prognostic factors, accurate prediction of risk fortreatment failure or relapse is not completely satisfactory.In order to improve risk assignment and develop new therapeuticstrategies, gene expression profiling and proteomicanalysis seem to offer important improvements in leukemiaclassification.

2006 - La risposta immunitaria verso il carcinoma prostatico: basi molecolari per nuove strategie terapeutiche [Partecipazione a progetti di ricerca]
F. Pagano; A. Viola; S. Bicciato; L. Matera

Il sistema immunitario possiede tutte le caratteristiche per rappresentare un'arma molto potente contro il cancro. Infatti, esso ècaratterizzato da un'elevata selettività che consente il riconoscimento specifico degli antigeni espressi dalle cellule tumorali e glieffettori principali dell'immunità acquisita, ovvero le cellule T, possiedono una straordinaria capacità di proliferare ed espandersi inrisposta alla stimolazione antigenica, quando questa sia associata agli opportuni segnali di costimolazione. Tutte queste proprietàhanno suscitato l'interesse di molti ricercatori verso approcci terapeutici immunologici, sia attivi (basati sull'induzione dellarisposta immune nello stesso ospite portatore di tumore) sia passivi (ovvero associati al trasferimento nel paziente di effettori dellarisposta immune previamente attivati in vitro). Tuttavia, nonostante le notevoli potenzialità, gli studi clinici pilota in cui sono statisperimentati nuovi protocolli immunoterapeutici hanno prodotto risultanti sconfortanti che indicano chiaramente come, allo statodell'arte, le conoscenze dei meccanismi immunologici non siano ancora sufficienti per delineare un protocollo terapeutico datrasferire alla clinica. Appare oramai chiaro che il microambiente tumorale è in grado di contrastare efficacemente l'azioneantitumorale degli effettori immunologici e che la definizione di un'efficace terapia immune può passare solo attraverso un'esaustivacomprensione dei circuiti soppressori che operano all'interfaccia ospite-tumore. Le cellule tumorali, infatti, si avvalgono di altrecellule, non solo per supportare la loro crescita locale, indurre la neovascolarizzazione, invadere i tessuti normali e metastatizzare adistanza, ma anche per creare un microambiente altamente sfavorevole all'azione dei linfociti. Un esempio importante di tale azionedi contrasto, emerso dagli studi dei gruppi afferenti al progetto, è costituito dall'alterazione del metabolismo di alcuni aminoacidiall'interno della massa neoplastica che ha rilevanti effetti negativi sull'attività antitumorale dei linfociti infiltranti il tumore. Questimeccanismi d'evasione tumorale costituiscono potenziali bersagli per lo sviluppo d'approcci immunoterapeutici innovativi. Ilpresente progetto si propone di identificare, validare e caratterizzare a livello molecolare nuovi approcci immunoterapeutici basatisulla combinazione di vaccinazione con antigeni prostatici tumore-associati e trattamento con composti che alterano lecaratteristiche immunosoppressive del microambiente creato dal tumore prostatico. L'approccio immunoterapeutico attivo saràcostituito dalla vaccinazione di pazienti affetti da cancro alla prostata con cellule dendritiche caricate con antigeni tumoraliprostatici e rappresenta uno dei primi trials clinici di questo tipo approvati nel nostro paese. In parallelo, verranno analizzate,avvalendosi anche di tecniche di genomica funzionale, le alterazioni indotte sui meccanismi di attivazione delle cellule Ttumore-specifiche da composti in grado di modificare il metabolismo intratumorale dell'arginina. Infatti, il gruppo proponente harecentemente dimostrato come la simultanena attivazione di due enzimi coinvolti nel metabolismo dell'aminoacido L-arginina(Arginasi e Sintetasi dell'ossido nitrico) sia in grado di generare, direttamente nel sito tumorale, segnali inibitori così potenti dainativare quasi completamente l'azione dei linfociti T tumore-specifici. Infine, verranno progettati e testati nuovi composti in gradodi alterare il metabolismo dell'arginina (o le cascate di segnalazione a monte) senza manifestare gli effetti di tossicità indotti daicomposti attualmente disponibili. La presenza nel gruppo proponente di due unità di Urologia assicurerà la disponibilità delmateriale biologico necessario agli studi sull'uomo. Gli obiettivi specifici del progetto sono: 1) valutare la fattibilità dellavaccinazione con cellule dendritiche in pazienti prece

2006 - Lymph node metastatic fingerprint revealed by genome-wide transcriptional profiling of primary breast cancer [Abstract in Rivista]

Breast cancer is the most frequent neoplasm and the leading cause of cancer mortality in women worldwide. Metastasis to the axillary lymph nodes through the lymphatic vessels is a common step in the progression of breast cancer and an important prognostic factor. Moreover, metastatic involvement is the basis for surgical treatment of axillary lymph nodes. In order to seek for gene expression patterns associated with lymph node metastasis in primary breast cancer, we compared the transcriptional profiles of 20 radically resected primary breast cancers from patients without lymph node metastasis with those of 19 primary breast cancers with lymph node metastasis using high-density oligonucleotide microarrays (Affymetrix). Transcriptional data indicates that samples with lymph node metastasis can be distinguished from patients without metastasis and constitute a cluster with a clearly characterized gene expression fingerprint. A supervised analysis of the 39 cases stratified into 2 classes based on lymph-nodal metastasis identified 46 differentially expressed probe sets. In particular, ANOVA technique allowed selecting only those probes correlated to metastasis correcting for the estrogen receptor factor. The group of metastatic samples resulted in a well defined entity showing a distinctive gene expression pattern characterized by the over-expression of 23 probes, representing genes mainly involved in cell adhesion. Conversely, cancers from patients without lymph node metastasis presented the over-expression of transcripts related to protein transport, processing, and metabolism.Our data provide insights into the molecular and biological features of breast cancer development and progression. In addition, the differentially expressed genes identified may represent prognostic molecular markers of potential clinical relevance.

2006 - Strategies for comparing gene expression profiles from different microarray platforms: Application to a case-control experiment [Articolo su rivista]

Meta-analysis of microarray data is increasingly important, considering both the availability of multiple platforms using disparatetechnologies and the accumulation in public repositories of data sets from diVerent laboratories. We addressed the issue of comparinggene expression proWles from two microarray platforms by devising a standardized investigative strategy. We tested this procedure bystudying MDA-MB-231 cells, which undergo apoptosis on treatment with resveratrol. Gene expression proWles were obtained using highdensity,short-oligonucleotide, single-color microarray platforms: GeneChip (AVymetrix) and CodeLink (Amersham). Interplatformanalyses were carried out on 8414 common transcripts represented on both platforms, as identiWed by LocusLink ID, representing 70.8%and 88.6% of annotated GeneChip and CodeLink features, respectively. We identiWed 105 diVerentially expressed genes (DEGs) on Code-Link and 42 DEGs on GeneChip. Among them, only 9 DEGs were commonly identiWed by both platforms. Multiple analyses (BLASTalignment of probes with target sequences, gene ontology, literature mining, and quantitative real-time PCR) permitted us to investigatethe factors contributing to the generation of platform-dependent results in single-color microarray experiments. An eVective approach tocross-platform comparison involves microarrays of similar technologies, samples prepared by identical methods, and a standardized batteryof bioinformatic and statistical analyses.

2006 - Toward the identification of a tolerogenic signature in IDO-competent dendritic cells [Articolo su rivista]

Although much is known on the transcriptional profiles of dendritic cells (DCs)during maturation, the molecular switches critical for the induction of a tolerogenicprogram in DC subsets are still obscure. We examined the gene expression profile ofmurine splenic CD8+ DCs rendered highly tolerogenic by interferon (IFN)-γ, whichactivates the enzyme indoleamine 2,3-dioxygenase (IDO, encoded by Indo) and thusinitiates the immunosuppressive pathway of tryptophan catabolism. By examining theexpression of a series of relevant genes in IDO+ versus IDO- DCs, we foundconsistent and selective association of the IDO-competent phenotype with downmodulationof the Tyrobp gene, encoding the signaling adapter DAP12, whichtypically associates with activating receptors. Down-modulation of Tyrobp involvedIFN consensus sequence binding protein (ICSBP), a transcription factor also knownas IRF-8. In both murine and human monocyte-derived DCs, silencing DAP12expression imparted IDO functional competence to IDO- cells, whereas silencing IRF-8 in IDO+ counterparts abolished IDO expression and function. Thus, IRF-8 isrequired in tolerogenic DCs for positive regulation of Indo and negative regulation ofTyrobp. Overall, these studies reveal the occurrence of a simple and evolutionarilyconserved code in the control of tolerance by an ancestral metabolic enzyme.

2006 - Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8(+) T cells [Articolo su rivista]

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(+) (CD 11b(+)IL-4R alpha(+)), inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-gamma released from T lymphocytes. CD11b(+)IL-4R alpha(+) 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.

2006 - Validation by RQ-PCR and flow cytometry of alpha-defensin1-3 (DEFA1-3) overexpression in relapsed and refractory acute lymphoblastic leukemia [Articolo su rivista]

In spite of high Cure rates and improved overall survival, 25% of pediatric patients with acute lyrnphoblastic 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 earlyrelapse (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-defensitil-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.

2005 - Analysis of diffuse large B cell lymphomas (DLBCL) transcriptional profiles at chromosomal level [Abstract in Rivista]

Introduction: Transcriptional profiling of whole genomes using cDNA or oligonucleotide high-density arrays is becoming increasingly popular among the biomedical research community. The efficient exploitation of gene expression databases requires not only computational tools for management, analysis, and functional annotation of primary data, but also integrating lists of modulated genes with of other sources of genomic information, such as gene sequence, locus or structural characteristics. In particular, integration between expression profiles and chromosomal localizations could be effective in detecting gene structural abnormalities such as genomic gains and losses and/or translocations. The aim of the present study is to apply bioinformatic tools for mapping transcriptional data at chromosomal level and detecting clusters of regionally modulated genes in DLBCL lymphoma datasets.Methods: A computational procedure has been designed to integrate expression profiles and gene sequence information for the identification of chromosomal regions with altered gene expression. The method is based on the application of a smoothing, coordinate-dependent function (e.g., cubic splines) to a standard transcriptional specificity statistic (e.g., standard F-statistic), commonly used to detect differentially expressed genes. Results: The analysis at chromosomal level of DLBCL transcriptional data presented by Shipp et al (Shipp et al., 2002) and Klein et al (Klein et al., 2001) allowed the detection of regional signals corresponding to known as well as putative loci with high frequent genomic losses and gains or marking translocation events: 1q, 3p, 6p, 9q, 11p, 1q, 12q, 17p, 17q, 18q, 19q. Data validated by real-time PCR on DLBCL samples will be presented Regions will also be correlated with survival data.Conclusions: The proposed analysis could help to identify genomic regions involved in the pathogenesis of DLBCL.

2005 - Analysis of gene expression profiles at chromosomal level [Abstract in Rivista]

Transcriptional profiling of whole genomes using cDNA or oligonucleotide high-density arrays is becoming increasingly popular among the biomedical research community. Although advances in technology and the rapid rise in microarray data availability are leading to new insight into fundamental biological problems, investigators are still confronted with the major problem of upgrading the information content of regulated gene lists obtained from microarray experiments. Indeed, the efficient exploitation of gene expression databases requires not only computational tools for management, analysis, and functional annotation of primary data, but also integrating lists of modulated genes with of other sources of genomic information, such as gene sequence, locus or structural characteristics. In particular, integration between expression profiles and chromosomal localizations could be effective in detecting gene structural abnormalities such as genomic gains and losses and/or translocations. The aim of the present study is to apply computational tools for mapping transcriptional data at chromosomal level and detecting clusters of regionally modulated genes in cancer specimens.Statistical tests and signal processing procedures are used to integrate expression profiles and gene sequence information and identify peculiar regions of modulated expression. In particular, the method is based on the application of a smoothing, coordinate-dependent function (e.g., cubic splines) to a standard transcriptional specificity statistic (e.g., standard F-statistic), commonly used to detect differentially expressed genes.This computational tool has been tested on different microarray data sets obtained from various human tumor samples (e.g., solid tumors and hematological disorders). In particular, the application of chromosomal level analysis to the transcriptional database presented by Bhattacharjee (Bhattacharjee et al., 2001), Armstrong (Armstrong et al., 2002), and Ross (Ross et al., 2003) allowed the detection of regional signals corresponding to known as well as putative loci with high frequent genomic losses and gains or marking translocation events.

2005 - Analysis of un-replicated time-course microarray experiments [Abstract in Rivista]

Since transcriptional control is the result of complex networks, analyzing dynamical states of gene expression is of paramount importance to detect the multivariate nature of biological mechanisms. Although hundreds of studies fully demonstrated the relevancy of microarrays in describing different physiological conditions, to reconstruct complex interaction pathways it is necessary to analyze the temporal evolution of transcriptional states. However, a robust experimental design for identifying differentially expressed genes over a temporal window would require large amounts of microarrays. Unfortunately, replicates for each time point and experimental condition are not always available, because of cost limitations and/or biological samples scarcity. In addition, common data analysis tools, like ANOVA, require replicates and disregard correlation structure among times. We present a method for the identification of differentially expressed genes in un-replicated time-course experiments. The procedure does not assume any model or distribution function, takes into account the correlation of data, and does not require sample replicates at the various time points, other than the presence of an initial time point for all analyzed conditions. The identification of differentially expressed genes as the result of a system perturbation is formally stated as a hypothesis testing problem in which a defined statistic is used to rank transcripts in order of evidence against the null hypothesis. Specifically, i) data are structured so that measurements are correlated in time, within the same biological condition; ii) the null hypothesis is formulated so that changes in expression levels at different time points are equivalent; iii) time point t0 represents the system before the perturbation. Therefore, modulated genes are detected testing the statistical significance of expression differences between physiological states at each time point, once corrected by the variability at t0, and given an empirical null distribution constructed using permutations. Statistical significance is assessed by the q-value. The method has been tested on time-course microarray experiments aimed at studying the temporal changes of gene expression in: i) skeletal muscle cells treated with a histone deacetylase inhibitor (Iezzi et al., Dev Cell; 2004) and ii) immature mouse dendritic cells (DC) exposed to larval and egg stages of S. mansoni (Trottein et al., J Immunol; 2004). Differentially expressed genes, identified using the proposed algorithm, have been compared with results obtained from ANOVA model and SAM paired test. The biological significance and soundness of selected transcripts was also verified using global functional profiling by means of OntoTools. Results demonstrate that this novel procedure allows the identification of biologically relevant genes using half of the replicates required by standard model-based approaches.

2005 - Different gene expression profiles of CD133+cells from cord blood and mobilized peripheral blood [Abstract in Rivista]

Cord blood (CB) is a valid alternative to mobilized peripheral blood (mPB) as a source of hematopoietic stem cell for clinical transplantation. The qualitative differences among CD133+ cells from these sources have been demonstrated by in vitro and in vivo studies but the molecular phenotype of these cells is only partially understood. In view of better exploiting the peculiarities and advantages of both the stem cell sources, we performed a comparative analysis of gene expression of CD133+ from CB and mPB using Affymetrix HG-U133A arrays. The CD133+ cells were selected using the MiniMacs immunomagnetic separation system (Miltenyi Biotec) from 6 CB units and 6 mPB samples. Since microarray studies are strictly dependent on the purity of the cell population and on the high RNA quality, it was crucial to apply rigorous quality controls. The purity of CD133+ cells was consistently more than 93% in all samples. In addition, after extraction, the RNA was purified by the RNeasy system (Qiagen) and the RNA quality was evaluated using the RNA 6000 Nano LabChip kit (Agilent Technologies). Given the low amount of RNA due to the limited numbers of CB CD133+ cells, we used a double-cycle amplification protocol for both sources to obtain biotin-labeled target for hybridization starting from as low as 100 ng of total RNA. Probe level data have been converted to expression values using Affymetrix MAS 5.0 algorithm. Differentially expressed genes have been identified using Significance Analysis of Microarrays (SAM). The cutoff for significance has been determined tuning the  parameter on the false positive rate and controlling the q-value for the gene list. We found that mPB CD133+ cells expressed cell cycle driving, transcription factor and DNA synthesis genes at distinctly higher levels than CB CD133+ cells, indicating a higher cycling activity in mPB CD133+ cells. Furthermore apoptosis driving genes were up-regulated in mPB CD133+ cells, probably due to G-CSF mobilization. Conversely in CB CD133+ cells we found an overexpression of genes involved in immunoresponse and immunomodulation. Since all the CB units were collected after vaginal delivery, this finding could be related to the stress status during labor. Finally, many genes related to different metabolic pathways such as alcohol, sterol, nucleotide, lipid and sugar biosynthesis, were found up-regulated in CB CD133+ cells, reflecting a neonatal wider potentiality of these cells.In conclusion, mPB CD133+ cells include a more committed and cycling population while CB CD133+ represent a cell subset with higher potentialities probably due to their immaturity. This suggest that even if CB has a lower number of stem cells, CB CD133+ cells are more prone/open/susceptible to different stimuli and potential fates.

2005 - Gene expression profiling of plasma cell dyscrasias reveals molecular patterns associated with distinct IGH translocations in multiple myeloma [Abstract in Rivista]

Introduction: Multiple Myeloma (MM) is characterized by genetic heterogeneity and varied clinical course. MM may develop from a premalignant condition called monoclonal gammopathy of undetermined significance (MGUS) and progress to plasma cell leukemia (PCL). Chromosomal translocations involving the immunoglobulin heavy-chain locus (IGH) and a promiscuous array of partner loci represent early and frequent genetic aberrations in MM.Methods: The gene expression profiles of purified plasma cells isolated from 7 MGUS, 39 MM and 6 PCL patients were analyzed by DNA microarrays, to investigate the molecular characterization of MM and the contribution of specific genetic lesions to the biological and clinical variability of MM.Results: MGUS patients could be distinguished from PCLs and the majority of MMs, while MMs were characterized by highly heterogeneous phenotype, being mainly grouped according to the presence of the most recurrent IGH translocations and specific gene expression signatures associated with each lesions. Overexpression of CCND2 and genes involved in cell-adhesion pathways in patients with dysregulated MAF and MAFB, upregulation of genes with apoptosis-related functions in t(4;14) patients and downregulation of the IL6-R in t(11;14) patients were identified. In addition, a group of MM patients with aggressive clinical evolution is characterized by a set of cancer germ line-specific antigens, a finding that could have implications for patients’ classification and immunotherapy in MM.Conclusions: Our study supports the notion of a marked heterogeneity of MM and provides insights into the role of distinct molecular pathways in MM.

2005 - Gene expression profiling of plasma cell dyscrasias reveals molecular patterns associated with distinct IGH translocations in multiple myeloma [Articolo su rivista]

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, 39MM 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 a-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.

Tagliafico, Enrico; Tenedini, Elena; Manfredini, Rossella; Ferrari, Sergio; Roncaglia, Enrica; Fantoni, Luca; Grande, Alexis; Parenti, Sandra; ZANOCCO MARANI, Tommaso; Gemelli, Claudia; Vignudelli, Tatiana; Montanari, Monica; Zini, Roberta; Salati, Simona; Bianchi, Elisa; Bicciato, Silvio; Specchia, Giorgina; Martinelli, Giovanni; Baccarani, Michele; Piccaluga, Pier Paolo; Torelli, Umberto; Ferrari, Sergio

Acute Myeloid Leukemia (AML) blast cells are immature committed myeloid cells unable to spontaneously undergo terminal maturation, characterized by heterogeneous sensitivity to natural differentiation inducers. No data are available so far by which infer the AML’s response to differentiating therapy. Thus, we have initially profiled by GeneChip arrays the gene expression of several AML cell lines: they derived by the original blast cell populations and are still characterized by the same immunophenotype, retain a different sensitivity or resistance to ATRA and VD and never undergo spontaneously terminal maturation. Here we show that differences exist by which predict the cell line differentiation fate. Next we constructed a signature able to predict resistance or sensitivity to the differentiation induction and tested it, using a TaqMan platform, for its capability to predict the in-vitro response of 28 VD or ATRA treated AML blast cell populations. Finally, by a meta-analysis of public available microarray data we demonstrated that our signature, that was formerly designed to identify differentiation therapy resistant populations, turned out to be a good classifier for clusters of patients with citogenetically and molecularly defined lesions that are known to have poor prognostic significance.

2005 - Molecular classification of multiple myeloma: A distinct transcriptional profile characterizes patients expressing CCND1 and negative for 14q32 translocations [Articolo su rivista]

Purpose 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.Results 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.Conclusion 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.

2005 - Molecular classification of multiple myeloma: A distinct transcriptional profile characterizes patients expressing CCND1 and negative for 14q32 translocations. [Abstract in Rivista]

The deregulation of CCND1, CCND2 and CCND3 genes represents a common event in multiple myeloma (MM), being at least one of them deregulated in almost all MM tumors. A recently proposed TC classification1 grouped MM patients into five classes on the basis of their cyclins D expression profiles and the presence of the main translocations involving the immunoglobulin heavy-chain (IGH) locus at 14q32. The aim of our study was to identify the putative transcriptional fingerprints associated with the deregulation of the different D-type cyclins and the presence of IGH translocations. The cyclin D expression levels obtained by high-density oligonucleotide microarray analysis of purified plasma cells from 50 MM cases were used to stratify the samples into the five TC classes, along with the molecular characteristics. The cyclin D expression data were validated by means of real-time quantitative PCR 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. A multi-class classification analysis was performed on the gene expression data and used to identify the transcriptional fingerprints of the 5 TC groups. 112 probe sets were selected as characterizing the TC1, TC2, TC4 and TC5 groups, whereas the TC3 samples showed heterogeneous phenotypes and no marker genes. In particular, TC1, TC4 and TC5 groups were characterized by the molecular signatures associated with the primary IGH translocations target genes. The TC2 group, showing significantly extra copies of the CCND1 locus (P=5.9×10-3) and neither IGH translocations nor the chromosome 13q deletion (P=1.7×10-3), was characterized by the overexpression of 30 genes, mainly involved in protein biosynthesis at translational level. Among the most specifically modulated transcripts within the group we identified a novel gene containing a BTB/POZ domain, typical of many zinc finger transcription factors and associated with transcriptional repression activity. A meta-analysis performed on two publicly available MM datasets, containing almost 250 cases, validated the identified gene expression signatures with a global classification rate (indicating the correct prediction of the TC class for the independent set) of 86% and 90%, respectively. Our data contribute to the understanding of the molecular and biological 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.

2005 - Molecular profiles associated with cyclins D dysregulation and IGH translocations in multiple myeloma [Abstract in Rivista]

Introduction: The deregulation of CCND1, CCND2 and CCND3 genes may represent a unifying oncogenic event in multiple myeloma (MM), being at least one of them deregulated in almost all MM tumors. A recent classification (TC classification) grouped MM patients into 5 classes on the basis of the Cyclin D expression profiles and the presence of the main translocations involving the immunoglobulin heavy chain locus (IGH) at 14q32.Methods: The gene expression profiles of purified plasma cells from 50 MM cases were used to stratify the samples into the 5 five TC classes and to identify the transcriptional fingerprints of these subgroups. Cyclins D expression data were validated by Q-RT-PCR analysis; FISH was used to investigate Cyclin D loci arrangement, to detect the main IGH translocations and the chromosome 13q deletion. Results: 110 probe set were identified by class-prediction analysis as characterizing TC1, TC2, TC4 and TC5 groups, while TC3 samples showed a heterogeneous phenotype and no marker gene. TC2 group, showing the presence of extracopies of CCND1 locus, was characterized by the overexpression of genes involved in protein biosynthesis at the translational level. A meta-analysis on published datasets validated the identified expression signatures. Conclusions: Our data provide contributions to the understanding of the molecular and biological features of distinct subtypes of MM. The identification of distinctive patterns of gene expression associated with TC2 patients may improve the risk stratification and indicate novel therapeutic targets.

2005 - Thalidomide downregulates angiogenic genes in bone marrow endothelial cells of patients with active multiple myeloma [Articolo su rivista]

PurposeTo study the antiangiogenic effect of thalidomide.Patients and MethodsThe 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.ResultsThalidomide 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.ConclusionInhibition by thalidomide is probably confined to the genes of active MMECs and KS. This would account for its higher efficacy in these diseases.

2004 - Acute Leukemia Subclassification: A Marker Protein Expression Perspective [Articolo su rivista]

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.

2004 - Analisi di dati di DNA microarray: fondamenti, selezione di geni, classificazione [Capitolo/Saggio]

Il sequenziamento del genoma e lo sviluppo parallelo di tecniche computazionali e di nuove tecnologie in biologia molecolare hanno permesso, in anni recenti, di intraprendere un’analisi sistematica dei meccanismi micromolecolari alla base dei sistemi biologici. In particolare, a partire dal '95, lo sviluppo della tecnologia delle matrici ad alta intesità, o microarray, di oligonucleotidi (Lockhart et al. 1996) e di cDNA (Schena et al. 1995) ha reso possibile estendere all’intero genoma la misura del livello di espressione, che con i Southern blot, introdotti nel 1975, era limitata ad un ridotto numero di geni preselezionati, dando così la possibilità di "fotografare" il livello di trascrizione di tutti i geni, in un dato istante, in un dato tessuto, in una specifica condizione fisiologica (Brown et al. 1999). Fisicamente un microarray è costituito da un supporto in vetro o silicio, su cui sono ancorate sequenze di nucleotidi specifiche per ciascun gene monitorato. L’RNA viene estratto dalle cellule e, dopo alcune reazioni biochimiche quali la trascrizione inversa e l’incorporazione di fluorescenti, viene ibridato all’array. Data la complementarietà specifica delle basi degli acidi nucleici, il numero di molecole che si ancorano in corrispondenza alla sequenza interrogante di un gene, determina l’intensità del fluorescente in quel punto. Le matrici ibridizzate vengono esposte ad una sorgente di luce laser e gli spettri di emissione vengono raccolti da uno scanner. Le immagini che così si ottengono indicano livelli diversi di espressione genica: in corrispondenza di ogni punto della matrice, la posizione individua il gene mentre l’intensità del segnale individua la quantità di RNA, quindi il suo livello di espressione. Attualmente sono presenti sul mercato due differenti tipi di microarray: gli spotted microarray, inizialmente sviluppati preso l'Università di Stanford, che misurano l’espressione genica relativa di una condizione sperimentale rispetto ad un’altra e i microarray ottenuti con tecniche fotolitografiche sviluppati e commercializzati da Affymetrix, i quali forniscono invece una misura di espressione genica assoluta. Mentre si rimanda a (Bicciato et al. 2000) per approfondimenti sugli aspetti tecnologici, qui è importante sottolineare che entrambe le tecniche, pur presentando aspetti peculiari che caratterizzano la loro realizzazione, la fase sperimentale e quella successiva di elaborazione del segnale, forniscono la misura parallela dell'espressione di migliaia di geni. Si intravede facilmente l'immenso potenziale che questa tecnologia offre, per rispondere a quesiti sia di tipo diagnostico/prognostico che di indagine funzionale. Nel primo caso l’obiettivo dell'analisi dei dati è la classificazione dei tessuti o delle malattie su base genetica, nel secondo caso è l'estrazione di informazioni che vanno dalla scoperta delle caratteristiche funzionali, strutturali o di regolazione di geni sconosciuti o solo parzialmente noti, all'individuazione di gruppi di geni co-espressi fino ad arrivare ad una analisi dei meccanismi di regolazione del processo trascrizionale e, in prospettiva, all’identificazione dei sistemi di controllo gene-gene che contribuiscono alla regolazione dei processi metabolici della cellula.

2004 - Analisi di dati di DNA microarray: reti di regolazione [Capitolo/Saggio]

I processi cellulari coinvolgono milioni di molecole che svolgono un ruolo coerente al fine di scambiare materia, energia e informazione con l’ambiente. Questi processi vengono regolati dai geni, la cui espressione è a loro volta regolata da una rete di interazioni fra altri geni, proteine e piccole molecole, detta rete di regolazione genica. Le reti di regolazione riguardano una svariata serie di segnali sia intracellulari, come le concentrazioni di mRNA, sia intercellulari, come gli ormoni, sia extracellulari, quali le variazioni delle condizioni ambientali (come gli stress termici). In maggior dettaglio, i geni vengono tradotti in proteine tramite un sistema di trascrizione (da DNA a mRNA) e traduzione (da mRNA a proteine) che è controllato da vari meccanismi (Nelson e Cox, 2002, Kohane et al., 2003). La trascrizione ha inizio in una particolare regione del DNA nota come regione del promotore, che è solitamente caratterizzata dalla presenza di segnali di riconoscimento per fattori di trascrizione, come la "TATA box" (TATAAA), "GC box", o "CAAT box" (CCAAT). Alla TATA box si lega una proteina, detta TBP (“TATA-binding” protein”), che, insieme ad altre proteine, consente all’enzima che permette la trascrizione, la RNA-polimerasi, di posizionarsi correttamente e venire attivata per iniziare la sintesi di RNA. I meccanismi di regolazione della trascrizione coinvolgono una serie di proteine, dette fattori di trascrizione, che possono bloccare il legame al promotore, o viceversa aumentare la probabilità che la RNA-polimerasi vi si leghi. In aggiunta a questi meccanismi, vi sono altri meccanismi di controllo che possono dar luogo, ad esempio, a splicing alternativi e modifiche post-trascrizionali. Tutti questi meccanismi influenzano la traduzione del messaggero in proteine. Per altro, proteine diverse possono essere formate a seguito dell’attivazione di un medesimo gene anche per effetto di modifiche post-traduzionali, anch’esse governate dalla presenza di proteine specifiche. La concentrazione di ogni proteina inoltre, è fortemente dipendente dalla presenza di altre molecole sia all’interno sia al di fuori della cellula, come i metaboliti o altre proteine; il ruolo svolto da queste molecole dipende da un complesso sistema noto come “signalling network”, che comprende recettori di membrana, proteine di “segnalazione” intra e intercellulari e i destinatari dei messaggi, ad esempio enzimi, altre proteine o complessi proteici (Roberts et al., 2000). E’ evidente quindi come lo studio delle reti di regolazione genica richieda di tener conto di aspetti genomici, proteomici e di regolazione metabolica. Non è perciò difficile apprezzare l’enorme complessità che queste reti possono raggiungere né la difficoltà di una loro modellizzazione matematica. La disponibilità dei DNA microarrays ha reso oggi possibile effettuare delle misure di espressione a livello dell’intero genoma e, grazie ai dati raccolti nel tempo, di osservarne la dinamica in diversi contesti sperimentali (Kohane et al., 2003). Grazie a queste osservazioni, è possibile inferire delle relazioni temporali fra le espressioni dei geni e, quindi, formulare delle ipotesi sulla struttura delle reti di regolazione. Questa attività è chiamata “reverse-engineering”, in quanto affronta il problema inverso di formulare delle ipotesi sul sistema dinamico che ha generato i dati sulla base dell’osservazione dei dati stessi (de Jong, 2002, Brazhnik et al., 2002).Nel corso degli ultimi anni sono stati proposti molti metodi per la generazione di reti di regolazione sulla base di dati di DNA microarrays (de Jong, 2002). Gli “ingredienti” della maggior parte di questi metodi sono una rete di interazioni, che esprime quali sono i legami fra i geni, ed un modello delle interazioni, in grado di descrivere la dinamica del sistema. Una rete di interazioni tra geni viene solitamente rappresentata trami

2004 - Artificial neural network technologies to identify biomarkers for therapeutic intervention [Articolo su rivista]

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.

2004 - Cancer genes involved in genetic progression of germinal centre B cell lymphomas [Partecipazione a progetti di ricerca]
F. Bertoni; T.C. Greiner; I. Kwee; S. Bicciato; M. Ponzoni; P.Y. Dietrich; G. Gaidano; G. Pruneri; M.G. Tibiletti

Non Hodgkin’s lymphomas (NHLs) represent the 6th most frequent cancer in men and women. Diffuse large B-cell lymphomas (DLBCLs) and follicular lymphomas (FLs) are the most common subtypes of all non-Hodgkin’s lymphomas. Despite they are considered indolent malignancies with a median overall survivall of approximately 10 years, FL represent an incurable and fatal disorder with nearly all patients dying of lymphoma, usually after histology transformation into the more aggressive DLBCL. Histological transformation occurs in 10-60% of FL independently from the type of chemotherapy.Both FL and DLBCL are characterised by specific cytogenetic anomalies such as t(14;18) involving the BCL2 gene and promiscuous rearrangements of BCL6 at 3q27. In addition these lymphomas may present a series of other different types of secondary genetic anomalies, mainly chromosomal changes but the clinical relevance of these anomalies is still unknown. The characterisation of the secondary chromosomal changes in both FL, DLBCL and in a selected series of transformed lymphomas may be helpful to understand the mechanisms of tumour progression and chemotherapy resistance and in addition may be relevant for cloning cancer genes involved in lymphomas transformation that are potential targets for gene-specific therapies.The applicant groups have characterised, with conventional and molecular cytogenetic approach, a series of 100 lymphomas including FL, DBLCL and a subset of serial samples from the same patients. Interestingly clonal secondary and specific chromosome changes have been identified involved both in transformation and in more aggressive lymphomas. In particular, chromosome rearrangements and deletions of 11q23->qter, 6q21->qter, 4p14->pter and 3p21->pter regions seems to be involved in the more aggressive and in the transformed lymphomas.The aim of this project is to characterise tumour genes mapping in the defined regions which contribute to the pathogenesis of DLBCL and to investigate the relationship between the expression profile of these genes and the clinical outcome.These goals will be carried out from the applicant groups using the following approaches:Genes identification in specific target regions (11q23->qter; 6q21->qter, 4p14->pter, 3p21) 1-Characterisation using conventional and molecular cytogenetics of new cases of lymphomas and a series of lymphoma cell lines in order to identify anomalies involving the target regions.2-Characterisation of retrospective cases of lymphomas and lymphomas cell lines using a more sensitive cytogenetic technology (Multicolor FISH, in situ hybridisation with specific PAC and BAC probes and arrayCGH) in order to refine at molecular level the previously identified abnormalities and to define the candidate molecular regions.3-Identification of candidate genes mapping in the candidate regions.Gene expression analysis1- Gene expression studies will be performed on the serial samples from the same patients, to look for the changes in expression profiles induced by the additional genetic abnormalities.2- Gene expression studies will be performed on a selected series of samples based upon the data discovered with arrayCGH, that is cases showing particular genomic losses and amplifications to be compared with cases with no abnormalities.3- Mathematical models will be designed and applied to predict the genomic profile, based upon the expression profile, as well to correlate genomic and expression data.4- Expression data will be validated using real-time PCR and whenever possible immunohistochemistry on a larger series of samples.Correlation between expression and clinical outcome and therapeutic approachesAll the genomic and expression data will be correlated to clinical and follow-up data to define factors that might be associated with genetic progression and worse clinical outcome.

2004 - Gene expression analysis of cord blood stem cells [Abstract in Rivista]

Cord blood (CB) is an attractive source for hematopoietic cellreplacement and CD34+ and CD133+ cells are the most clinically relevantcell subsets used for transplantation purposes. In view of characterizing thegene profile of these two populations of hematopoietic stem cells (HSCs), agenome-wide gene expression analysis is required. A powerful tool tobroaden the knowledge of molecular mechanisms of HSCs is geneexpression analysis using microarray technology. In this study wemeasured gene expression profiles of CD34+ and CD133+ CB cells usingAffymetrix HG-U133A arrays containing 22.000 probes. We processed 10CB units: CD34+ (n=5) and CD133+ (n=5) cells were selected using theMiniMacs immunomagnetic separation system (Miltenyi Biotec). Weevaluated RNA quantity and quality using the RNA 6000 Nano LabChipkit (Agilent Technologies). Given the low amount of RNA due to thelimiting numbers of HSCs obtained, we used a double-cycle amplificationprotocol to obtain biotin-labeled target for hybridization starting from aslow as 100 ng of total RNA. Gene expression profiling data were analyzedby means of two different standard software packages (MAS 5.0 anddChip). A supervised analysis allowed us to identify genes distinguishingthe two different groups of samples, one containing the CB CD34+ and theother containing the CB CD133+ samples. The comparison showed theover-expression of genes involved in cell homing (IL-8, CXCL4, CXCL7,fibronectin 1, collagen type IV) and myeloid differentiation (GMCSFR,HDC, MNDA, MRP) in the CD34+ subset. Moreover, no gene was foundto be up-regulated in CD133+ subset. These preliminary data on the geneexpression profiles of these two populations support the concept thatCD34+ cells represent a more committed, hematopoietic cell subsetcompared to CD133+ cells. In conclusion, our study may serve as a basisfor further researches in the field of functional genomic of HSCs.

2004 - Gene expression profiling of plasma cell dyscrasias: The role of IGH translocations in the heterogeneity of multiple myeloma. [Abstract in Rivista]

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 ofundetermined significance, MGUS) or progress from intra-medullary to extra-medullaryforms (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 analyzed the gene expression profiles of plasma cells isolated from 7 MGUS, 39 MM and 6 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 signatures 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. In addition, we identified a set of cancer germ-line antigens specifically expressed in a sub-group of MM patients characterized by an aggressive clinical evolution, a finding that could have implications for patient classification and immunotherapy.

2004 - Marker identification and classification of cancer types using gene expression data and SIMCA [Articolo su rivista]

Objectives. High-throughput technologies are radically boosting the understanding of living systems, thus creating enormous opportunities to elucidate the biological processes of cells in different physiological states. In particular, the application of DNA microarrays to monitor expression profiles from tumor cells is improving cancer analysis to levels that classical methods have been unable to reach. However, molecular diagnostics based on expression profiling requires addressing computational issues as the overwhelming number of variables and the complex, multi-class nature of tumor samples. Thus, the objective of the present research has been the development of a computational procedure for feature extraction and classification of gene expression data.Methods. The Soft Independent Modeling of Class Analogy (SIMCA) approach has been implemented in a data mining scheme, which allows the identification of those genes that are most likely to confer robust and accurate classification of samples from multiple tumor types.Results: The proposed method has been tested on two different microarray data sets, namely Golub's analysis of acute human leukemia [1] and the small round blue cell tumors study presented by Khan et al. [2]. The identified features represent a rational and dimensionally reduced base for understanding the biology of diseases, defining targets of therapeutic intervention, and developing diagnostic tools for classification of pathological states.Conclusions: The analysis of the SIMCA model residuals allows the identification of specific phenotype markers. At the some time, the class analogy approach provides the assignment to multiple classes, such as different pathological conditions or tissue samples, for previously unseen instances.

2003 - Computational analysis of flow-cytometry antigen expression profiles in childhood acute lymphoblastic leukemia: an MLL/AF4 identification [Articolo su rivista]

Precursor B-acute lymphoblastic leukemia (pB-ALL) is a heterogeneous disease and multiparameter flow cytometry, molecular genetics, and cytogenetic studies have all contributed to classification of subgroups with prognostic significance. Recently, gene expression microarray technology has been used to investigate lymphoblastic leukemias, demonstrating that known and novel pB-ALL subclasses can be separated on the basis of gene expression profiles. The strength of microarray technique lays in part in the multivariate nature of the expression data. We propose a parallel multiparametric approach based on immunophenotypic flow-cytometry expression data for the analysis of leukemia patients. Specifically, we tested the potential of this approach on a data set of 145 samples of pediatric pB-ALL that included 46 samples positive for mixed lineage leukemia (MLL) translocations (MLL+) and 99 control pB-ALLs, negative for this translocation ( MLL-). The expression levels of 16 marker proteins have been monitored by four-color flow cytometry using a standardized diagnostic panel of antibodies. The protein expression database has been then analyzed using those univariate and multivariate computational techniques normally applied to mine and model large microarray data sets. Marker protein expression profiling not only allowed separating pB-ALL cases with an MLL rearrangement from other ALLs, but also demonstrates that MLL+ leukemias constitute a heterogeneous group in which MLL/ AF4 leukemias represent a homogenous subclass described by a specific expression fingerprint.

2003 - PCA disjoint models for multiclass cancer analysis using gene expression data [Articolo su rivista]

Motivation: Microarray expression profiling appears particularly promising for a deeper understanding of cancer biology and to identify molecular signatures supporting the histological classification schemes of neoplastic specimens. However, molecular diagnostics based on microarray data presents major challenges due to the overwhelming number of variables and the complex, multiclass nature of tumor samples. Thus, the development of marker selection methods, that allow the identification of those genes that are most likely to confer high classification accuracy of multiple tumor types, and of multiclass classification schemes is of paramount importance.Results: A computational procedure for marker identification and for classification of multiclass gene expression data through the application of disjoint principal component models is described. The identified features represent a rational and dimensionally reduced base for understanding the basic biology of diseases, defining targets for therapeutic intervention, and developing diagnostic tools for the identification and classification of multiple pathological states. The method has been tested on different microarray data sets obtained from various human tumor samples. The results demonstrate that this procedure allows the identification of specific phenotype markers and can classify previously unseen instances in the presence of multiple classes.

2003 - Pattern identification and classification in gene expression data using an autoassociative neural network model [Articolo su rivista]

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 clatasets-Golub's analysis of acute human leukemia [Golub et al. (1999) Science 286:531537], 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.

2003 - Plasminogen activator inhibitor-2: A molecular biomarker for head and neck cancer progression [Articolo su rivista]

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.

2003 - Strategies and tools for upgrading the information content of regulated gene lists from microarray experiments [Articolo su rivista]

The transcriptional profiling of whole genomes using cDNA or oligonucleotide high-density arrays is becoming increasingly popular among the biomedical research community. Although advances in technology and the rapid rise in microarray data availability are leading to new insight into fundamental biological problems, investigators are still confronted with the major problem of upgrading the information content of regulated gene lists obtained from microarray experiments. indeed, the efficient exploitation of gene expression databases requires not only computational tools for management and analysis of primary data, but also integrating lists of modulated genes with of other sources of biological information, such as biochemical pathways and biomedical literature. However, such integration, essential to functionally interpret derived gene lists and to relate expression profiles to molecular physiology, often results in an explosion of the information content instead of knowledge growth. This article reviews several public resources for visualization, annotation, and functional interpretation of gene expression datasets. The focus is on tools that integrate functional genomic information, biochemical pathways, and bibliographic records with intuitive graphical summaries.

2003 - Synthetic pepticles derived from the angiostatin K4 domain inhibit endothelial cell migration [Articolo su rivista]

Angiogenesis, the process by which new capillaries are formedfrom preexisting blood vessels, represents a vital function for thegrowth of normal tissues during embryogenesis as well as for thepathological growth of tumors. Experimental evidence hasshown that both primary and metastatic tumors need to recruitangiogenic vessels for their growth, and form their owncirculatory system by up-regulating angiogenic stimulators anddown-regulating angiogenesis inhibitors.[1] Blocking of positiveregulators or utilization of negative regulators to suppressangiogenesis results in a delay or regression of induced tumors.In particular, negative regulators of angiogenesis, such asangiostatin, endostatin, and antagonists for integrin v3,displayed profound antitumor activities in vivo.[2]

2002 - Disjoint PCA models for marker identification and classification of cancer types using gene expression data [Articolo su rivista]

Background The parallel monitoring of the expression profiles of thousands of genes seems particularly promising for a deeper understanding of cancer biology and to identify molecular signatures supporting the histological classification schemes of neoplastic specimens. However, molecular diagnostic based on microarray data presents major challenges due to the complex, multiclass nature and to the overwhelming number of variables characterizing gene expression databases of multiple tumor samples. Thus, the development of multiclass; classification schemes and of marker selection methods, that allow the simultaneous classification of multiple tumor types and the identification of those genes that are most likely to confer high classification accuracy, is of paramount importance.Methods. A computational procedure for marker identification and classification of multiclass gene expression data through the application of disjoint principal component models, based on the Soft independent Modeling of Class Analogy approach (SIMCA), is described. The identified features represent a rational and dimensionally reduced base for understanding the basic biology of diseases, defining targets of therapeutic intervention, and developing diagnostic tools for the identification and classification of multiple pathological states.Results. The method has been tested on 2 different microarray data sets obtained from various human tumor samples: i) acute leukemias, and ii) small round blue-cell tumors.Conclusions. The results demonstrate that the disjoint PCA modeling procedure allows the identification of specific phenotype markers and provides the assignment to multiple classes for previously unseen instances.

2002 - Fermentation diagnosis by multivariate statistical analysis [Articolo su rivista]

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).

2002 - Prediction of in vivo synergistic activity of antiangiogenic compounds by gene expression profiling [Articolo su rivista]

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 thrombospondinmimetic 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.

2002 - Protein expression profiling of acute lymphoblastic leukemia subclasses. [Abstract in Rivista]

Parallel to fingerprinting of leukemia subclasses by gene expression profiling we propose to fingerprint leukemia subclasses using protein expression data collected by quantitative flow cytometry. A number of acute lymphoblastic leukemia subclasses with characteristic chromosomal aberrations (MLL; Bcr/Abl;E2A/PBX;TEL/AML1;hyperdiploid) have a unique gene expression profile as demonstrated by microarray studies (1-2). Among genes differentially expressed between leukemia subtypes are known proteins of which expression can be measured by quantitative flow cytometry using monoclonal antibodies (3). Here, univariate t-tests and multivariate principal component analysis (PCA) have been applied to profile phenotypes of 145 pB-ALL samples using expression data of 16 proteins. Expression of each marker protein has been quantified in terms of soluble fluorochrome (MESF) and coefficient of variation (CV). Materials:Patients material: A retrospective analysis has been performed on a total of 145 children diagnosed as suffering from precursor-B acute lymphoblastic leukemia (pB-ALL). We included 46 patients that were diagnosed for a MLL rearrangement between May 1995 and June 2001. As a control group representative of pB-ALL’s negative for MLL, we randomly selected 99 consecutively diagnosed patients from our MLL negative reference database.Among MLL patients, 26 are infants (aged < 1 year at diagnosis) and 20 non-infants while in the control group 5 infants (aged < 1 year at diagnosis) and 94 non-infants patients are present.

2001 - Identifying and discriminating seismic patterns leading flank eruptions at Mt. Etna Volcano during 1981-1996 [Articolo su rivista]

Seismicity affecting Mt. Etna Volcano (Italy) has been investigated in order to identify and discriminate seismic patterns precursory to flank eruptions. An intense period (1981-1996) of seismicity and volcanism, during which eight flank eruptions occurred has been considered. Two statistical methods are used: mean hypothesis testing and entropic decision trees. The results of the two methods are consistent and reveal a pattern of 'deep' and 'western' events, prior to the flank eruptions that can be used as a predictive tool as well as a physical modeling constraint.

2000 - Matrici di DNA per lo studio dell’espressione genica [Capitolo/Saggio]

Le ricerche svolte negli ultimi anni nel campo della biologia molecolare e della genetica hanno reso possibile lo sviluppo e la commercializzazione di una serie di tecnologie per l’analisi delle molecole costituenti gli organismi viventi. In particolare, la messa a punto delle tecniche di amplificazione e sequenziamento del DNA ha aperto la strada ai progetti di mappatura del genoma, tanto che, a tutt’oggi, si conosce l’intera sequenza genica di circa 25 microrganismi e si presume che già nei prossimi mesi sarà disponibile una prima stesura della sequenza genetica dell’uomo. Il risultato di questi progetti è stata la creazione di enormi banche dati che contengono la sequenza di tutti i geni di un particolare organismo e che possono essere utilizzate come vere e proprie mappe utili per lo studio e la comprensione dei meccanismi cellulari. Questa immensa quantità di informazioni rappresenta una delle sfide future delle scienze biologiche, mediche e biotecnologiche in quanto potrà consentire lo sfruttamento della intera sequenza primaria del DNA per l’identificazione delle regioni codificanti e regolatrici, delle variazioni polimorfiche tra le varie specie di organismi, dei meccanismi di sviluppo e degenerazione cellulare e dei sistemi di controllo e di interazione tra le diverse macromolecole biologiche. La complessità e la quantità di dati generati dai diversi progetti genoma pongono tuttavia serie limitazioni al tradizionale approccio riduzionista dell’analisi e dello studio di un gene o di una reazione alla volta e richiedono lo sviluppo di metodiche che permettano l’analisi simultanea di tutti i componenti di un sistema biologico [Lander, 1999]. Le matrici ad alta densità di DNA o di oligonucleotidi (DNA or oligonucleotide microarrays) rappresentano un metodo per esaminare in maniera simultanea e sistematica le variazioni del DNA e dell’RNA all’interno di un organismo e promettono di diventare uno strumento di uso comune nella ricerca medica e biologica. Con i microarray è infatti possibile monitorare i livelli di espressione in maniera sia statica che dinamica, ottenendo, simultaneamente e parallelamente, informazioni sia sulla localizzazione che sulle interazioni dell’espressione di un gene relativamente a quella di tutti gli altri.L’utilizzo delle matrici ad alta densità consentirà di aumentare enormemente la capacità di comprendere la funzione e la regolazione dei geni individuati grazie ai progetti di sequenziamento, purché siano parallelamente sviluppati dei metodi efficaci che consentono di sfruttare l’enorme massa di dati generati da queste tecnologie.

2000 - Mining of Biological Data I: Identifying Discriminating Features via Mean Hypothesis Testing [Articolo su rivista]

Large volumes of data are routinely collected during bioprocessoperations and, more recently, in basic biological research usinggenomics-based technologies. While these data often lack sufficientdetail to be used for mechanism identification, it is possible that theunderlying mechanisms affecting cell phenotype or process outcomeare reflected as specific patterns in the overall or temporal sensorlogs. This raises the possibility of identifying outcome-specificfingerprints that can be used for process or phenotype classificationand the identification of discriminating characteristics, such asspecific genes or process variables. The aim of this work is to providea systematic approach to identifying and modeling patterns inhistorical records and using this information for process classifica-tion. This approach differs from others in that emphasis is placed onanalyzing the data structure first and thereby extracting potentiallyrelevant features prior to model creation. The initial step in this over-all approach is to first identify the discriminating features of the rele-vant measurements and time windows, which can then be subse-quently used to discriminate among different classes of processbehavior. This is achieved via a mean hypothesis testing algorithm.Next, the homogeneity of the multivariate data in each class isexplored via a novel cluster analysis technique called PC1 TimeSeries Clustering to ensure that the data subsets used accuratelyreflect the variability displayed in the historical records. This will bethe topic of the second paper in this series. We present here themethod for identifying discriminating features in data via meanhypothesis testing along with results from the analysis of case studiesfrom industrial fermentations.

2000 - Mining of Biological Data II: Assessing Data Structure and Class Homogeneity by Cluster Analysis [Articolo su rivista]

An important step in data analysis is class assignment which isusually done on the basis of a macroscopic phenotypic or bioprocesscharacteristic, such as high vs low growth, healthy vs diseased state,or high vs low productivity. Unfortunately, such an assignment maylump together samples, which when derived from a more detailedphenotypic or bioprocess description are dissimilar, giving rise tomodels of lower quality and predictive power. In this paper we pre-sent a clustering algorithm for data preprocessing which involves theidentification of fundamentally similar lots on the basis of the extentof similarity among the system variables. The algorithm combinesaspects of cluster analysis and principal component analysis byapplying agglomerative clustering methods to the first principalcomponent of the system data matrix. As part of a rational strategyfor developing empirical models, this technique selects lots (sam-ples) which are most appropriate for inclusion in a training set byanalyzing multivariate data homogeneity. Samples with similar datastructures are identified and grouped together into distinct clusters.This knowledge is used in the formation of potential training sets.Additionally, this technique can identify atypical lots, i.e., samplesthat are not simply outliers but exhibit the general properties of oneclass but have been given the assignment of the other. The method ispresented along with examples from its application to fermentationdata sets.

2000 - Temporal Sequence Pattern Learning and Dynamic System Control (DSC) [Relazione in Atti di Convegno]

We have investigated the role of temporal sequence learning, using an unsuper- vised artificial neural network (1), called Monoconnected Autoreflexive Neural Net- work, for better understanding the implicit learning process role, involved during elementary associative learning processes. Several neural network models have been proposed to describe implicit learning (IL), using unsupervised and self-organized models (2, 3). In our experiments we used a real biochemical data set consisting of 15 features, that deals with penicillin production (112 temporal sequence blocks with 11 sequence points per block (1232 patterns). The prediction task requires that the neural network can predict the correct sequence position, after a preliminary training (50% of all patterns). After training, the neural network learn to find the correct position in the temporal sequences with good accuracy. Our results seem to confirm that elementary associative learning, could be used in temporal sequence learning and that dynamic system control (DSC) tasks (for instance to know which features are more sensitive to a better penicillin production), could be derived from the implicit learning process, using the importance of different features, recovered from the weight matrix analysis.

1999 - Identifying Seismicity Patterns Leading Flank Eruptions at Mt. Etna Volcano During 1981-1996 [Articolo su rivista]

Seismicity time properties of the Etna Volcano (Italy) are investigated through a systematic pattern recognition analysis. Mean Hypothesis Testing (MHT) has been applied to a long time database of instrumental data recorded from 1981 to 1996 to identify relevant correlation among seismic patterns, main seismogenic volumes and the eight flank eruptions occured in the same period. Time evolution of the test statistic (T-Ratio), calculated on a temporal window starting 75 days prior to a volcanic eruption and extending 25 days after, reveals that there is a pattern of seismic activity prior to the eruptions that can be used as a diagnostic tool as well as a physical modeling support in eruption forecasting

1998 - Automation of the Liquid-Phase Synthesis of Biopolymers [Articolo su rivista]

The liquid-phase synthesis of biopolymers is an alternative to the solid-phase methodologies that are widely applied in spite of limitations in the use of heterogeneous media. The advantages of the liquid-phase approach are due to the fact that the support is alternatively soluble in the solvents used for the chemical reactions and insoluble in the mixtures used for the purification steps. Thus, the advantages of both 'solution' and 'solid-phase' methodologies can be properly integrated. At present the liquid-phase procedure, unlike solid-phase methods that have been highly automated, can be carried out only manually due to technical difficulties imposed by the cyclic repetition of dissolution and precipitation steps. The original apparatus proposed here performs, for the first time, all the steps required by the liquid-phase approach in a fully automatic way controlled by a user-friendly dedicated software. The automated liquid-phase methodology has been tested by synthesizing some sample oligonucleotides and the preliminary results are discussed in the present paper.

1997 - A Novel Algorithm for the Coupling Control in Solid Phase Peptide Synthesis [Articolo su rivista]

This paper describes a new method for the evaluation of conductimetric data collected during the in-line monitoring of the coupling step in solid-phase peptide synthesis. The control scheme relies on a feed-forward artificial neural network algorithm which can predict the final yield of the reaction within its initial 5 min by analyzing the conductivity signal profile. The yield values predicted by the artificial neural network algorithm result in good accordance with the data obtained by the commonly used ninhydrin test.

1997 - SPPS of Difficult Sequences: a Comparison of Chemical Conditions, Synthetic Strategies and On-line Monitoring [Articolo su rivista]

The H-Ala-Arg-(Ala)(6)-Lys-OH sequence is a biologically interesting 'difficult sequence' presenting N-alpha-Fmoc deprotection and coupling problems. Different chemical conditions and synthetic strategies have been tested in order to overcome the problems due to sequence-dependent interactions. In particular, it was confirmed that different solvents in the deprotection step did not provide any significant improvement, but the use of a more efficient base in the deprotection mixture avoided insufficient unblocking of N alpha-protecting group; problems due to partial coupling in the last steps of the synthesis were solved by double coupling techniques. Moreover, the synthesis of the model peptide was carried out using both 'continuous flow' and 'batch' techniques. The present results demontrate that on-line monitoring of the deprotection step by absorbance measurements represents a very effective tool to detect the onset of internal aggregations during the synthesis

1995 - An Improved System for Automated Peptides Synthesis [Articolo su rivista]

This paper describes an original system for automated solid-phase peptide synthesis (SPPS) that was developed in our laboratories. The synthesizer is equipped with separate activator and batch reactors, and was designed to operate under either manual or computer control. Two metering pumps, which constitute the core of the entire apparatus, provide for the delivery and transfer of reagents and solvents in the appropriate quantities and in the correct sequence, and permit simultaneous condensation and activation operations that result in considerable time saving. Various applications to the synthesis of biologically important peptides, utilizing the most widely used strategies in SPPS, are presented as examples of this improved technology.

1994 - A Batch-Type System for Large-Scale Solid-Phase Oligonucleotide Synthesis [Articolo su rivista]

Synthetic biopolymers (e.g., peptides, oligosaccarides and oligonucleotides) are being increasingly utilized for human, animal, plant and food applications. In particular, the use of synthetic nucleic acid derivatives with potent and specific biological activities as diagnostic and therapeutic agents has recently attracted a lot of attention. In response to the synthetic needs connected with these applications, we have developed an automated batch-type system for oligonucleotide synthesis. The most characteristic features of this apparatus are a highly flexible control and a specially designed delivery system, both of which are compatible with the synthetic procedures and chemistries most used at present; they also permit an easy up-scaling of the reactions, and are readily adaptable to future improvements

1993 - Development of a Prototype for the Automated Solid-Phase Synhtesis of Peptides [Articolo su rivista]

Following the discovery of naturally occurring biopolymers with potent and/or specific biological activities, the past few years have seen an extraordinary progress in the chemical synthesis of molecules such as peptides, small proteins and oligonucleotides. The use of synthetic biopolymers in pharmacological and biomedical settings and, in particular, in human, animal, plant and food applications, requires large amounts of product. In response to these new synthetic needs, we are developing synthesizer prototypes for automated solid-phase synthesis of peptides and oligonucleotides