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Giulia P.,Polytechnic University of Turin | Acquaviva A.,Polytechnic University of Turin | Petti C.,Institute for Cancer Research at Candiolo IRCC | Isella C.,Institute for Cancer Research at Candiolo IRCC | And 3 more authors.
BIOINFORMATICS 2014 - 5th Int. Conf. on Bioinformatics Models, Methods and Algorithms, Proceedings; Part of 7th Int. Joint Conference on Biomedical Engineering Systems and Technologies, BIOSTEC 2014 | Year: 2014

Metastatic spread to the liver is a frequent complication of colorectal cancer (CRC), occurring in almost half of the cases, for which personalized treatment strategies are highly desirable. To this aim, it has been proven that patient-derived mouse xenografts (PDX) of liver-metastatic CRC can be used to discover new therapeutic targets and determinants of drug resistance. To identify gene fusions in RNA-Seq data obtained from such PDX samples, we propose a novel pipeline that tackles the following issues: (i) discriminating human from murine RNA, to filter out transcripts contributed by the mouse stroma that supports the PDX; (ii) increasing sensitivity in case of suboptimal RNA-Seq coverage; (iii) prioritizing the detected chimeric transcripts by molecular features of the fusion and by functional relevance of the involved genes; (iv) providing appropriate sequence information for subsequent validation of the identified fusions. The pipeline, built on top of Chimerascan(R.Iyer, 2011) and deFuse(McPherson, 2011) aligner tools, was successfully applied to RNASeq data from 11 PDX samples. Among the 299 fusion genes identified by the aforementioned softwares, five were selected since passed all the filtering stages implemented into the proposed pipeline resulting as biologically relevant fusions. Three of them were experimentally confirmed. Copyright © 2014 SCITEPRESS - Science and Technology Publications. All rights reserved. Source


Zanivan S.,Max Planck Institute of Biochemistry | Zanivan S.,Beatson Institute for Cancer Research | Maione F.,Institute for Cancer Research at Candiolo IRCC | Hein M.Y.,Max Planck Institute of Biochemistry | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2013

Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell-extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Lustgarten D.E.S.,Pulmonary Associates | Deshpande C.,University of Pennsylvania | Aggarwal C.,University of Pennsylvania | Wang L.-C.,University of Pennsylvania | And 12 more authors.
Journal of Thoracic Oncology | Year: 2013

PURPOSE: Thymidylate synthase (TS) is a potential predictor of outcome after pemetrexed (Pem) in patients with malignant pleural mesothelioma (MPM), and assays measuring TS levels are commercially marketed. The goal of this study was to further evaluate the value of TS and to study another potential biomarker of response, the enzyme, folyl-polyglutamate synthase (FPGS), which activates Pem intracellularly. METHODS: Levels of TS and FPGS were semi-quantitatively determined immunohistochemically using H-scores on tissue samples from 85 MPM patients receiving Pem as primary therapy. H-score was correlated with radiographic disease control rate (DCR), time to progression (TTP) and overall survival (OS). In addition, expression levels of TS and FPGS in MPM cell lines were determined using immunoblotting and correlated with their sensitivity to Pem-induced cell death. RESULTS: H-scores from patients with disease control versus progressive disease showed extensive overlap. There were no significant correlations of DCR, TTP, or OS to either TS levels (p = 0.73, 0.93, and 0.59, respectively), FPGS levels (p = 0.95, 0.77, and 0.43, respectively) or the ratio of FPGS/TS using the median scores of each test as cutoffs. There was no correlation between TS or FPGS expression and chemosensitivity of mesothelioma cells to Pem in vitro. CONCLUSIONS: Although previous retrospective data suggest that TS and FPGS expression might be potential markers of Pem efficacy in MPM, our data indicate these markers lack sufficient predictive value in individual patients and should not be used to guide therapeutic decisions in the absence of prospective studies. Copyright © 2013 by the International Association for the Study of Lung. Source


Bardella C.,University of Turin | Bardella C.,Institute for Cancer Research at Candiolo IRCC | Bardella C.,University of Oxford | Olivero M.,University of Turin | And 12 more authors.
Molecular and Cellular Biology | Year: 2012

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer. © 2012, American Society for Microbiology. Source


Luraghi P.,Institute for Cancer Research at Candiolo IRCC | Luraghi P.,University of Turin | Reato G.,Institute for Cancer Research at Candiolo IRCC | Reato G.,University of Turin | And 23 more authors.
Cancer Research | Year: 2014

Metastatic colorectal cancer remains largely incurable, although in a subset of patients, survival is prolonged by new targeting agents such as anti-EGF receptor (anti-EGFR) antibodies. This disease is believed to be supported by a subpopulation of stem-like cells termed colon cancer initiating cell (CCIC), which may also confer therapeutic resistance. However, how CCICs respond to EGFR inhibition has not been fully characterized. To explore this question, we systematically generated CCICs through spheroid cultures of patient-derived xenografts of metastatic colorectal cancer. These cultures, termed "xenospheres," were capable of long-term self-propagation in vitro and phenocopied the original patient tumors in vivo, thus operationally defining CCICs. Xenosphere CCICs retained the genetic determinants for EGFR therapeutic response in vitro and in xenografts; like the original tumors, xenospheres harboring a mutated KRAS gene were resistant to EGFR therapy, whereas those harboring wild-type RAS pathway genes (RASwt) were sensitive. Notably, the effects of EGFR inhibition in sensitive CCICs could be counteracted by cytokines secreted by cancer-associated fibroblasts. In particular, we found that the MET receptor ligand hepatocyte growth factor (HGF) was especially active in supporting in vitro CCIC proliferation and resistance to EGFR inhibition. Ectopic production of human HGF in CCIC xenografts rendered the xenografts susceptible to MET inhibition, which sensitized the response to EGFR therapy. By showing that RASwt CCICs rely on both EGFR and MET signaling, our results offer a strong preclinical proof-of-concept for concurrent targeting of these two pathways in the clinical setting. © 2014 American Association for Cancer Research. Source

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