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Dey J.H.,Friedrich Miescher Institute for Biomedical Research | Bianchi F.,Fondazione Instituto Firc Of Oncologia Molecolare | Voshol J.,Novartis | Bonenfant D.,Novartis | And 2 more authors.
Cancer Research | Year: 2010

Members of the fibroblast growth factor receptor (FGFR) family have essential roles in normal physiology and in cancer where they control diverse processes. FGFRs have been associated with breast cancer development. Thus, models to study the role of FGFR in breast cancer and their targeting potential are important. We present an in vitro and in vivo analysis of FGFRs in the breast cancer model cell lines 67NR and 4T1. We show that both tumor cell lines coexpress FGFRs and ligands and display autocrine FGFR signaling activity. Fibroblast growth factor receptor substrate 2 (FRS2), a downstream mediator of FGFR, is constitutively tyrosine phosphorylated and multiple signaling pathways are active. Treatment of 67NR and 4T1 cultures with TKI258, an FGFR tyrosine kinase inhibitor (TKI), caused a rapid decrease in FRS2 phosphorylation; decreased the activity of extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and phospholipase Cγ; and blocked proliferation of both tumor lines. Furthermore, TKI258 induced 4T1 apoptotic cell death via blockade of the phosphoinositide 3-kinase/AKT pathway. In vivo, one dose of TKI258 rapidly lowered FRS2 phosphorylation and ERK1/2 and AKT activity in mammary tumors. Long-term TKI258 treatment of 4T1 tumor- and 67NR tumor-bearing mice had a significant effect on primary tumor outgrowth and 4T1 tumor-induced lung metastases. A microarray analysis was carried out to identify targets with roles in TKI258 antitumor activity and potential prognostic markers in human breast tumors. Of interest are the downregulated matrix metalloproteases (MMP), in particular MMP9, which is essential for metastatic spread of 4T1 tumors. ©2010 AACR.


Maspero E.,Fondazione Instituto Firc Of Oncologia Molecolare | Valentini E.,Fondazione Instituto Firc Of Oncologia Molecolare | Mari S.,Fondazione Instituto Firc Of Oncologia Molecolare | Cecatiello V.,Italian National Cancer Institute | And 4 more authors.
Nature Structural and Molecular Biology | Year: 2013

Homologous to E6-AP C terminus (HECT) E3 ligases recognize and directly catalyze ligation of ubiquitin (Ub) to their substrates. Molecular details of this process remain unknown. We report the first structure, to our knowledge, of a Ub-loaded E3, the human neural precursor cell-expressed developmentally downregulated protein 4 (Nedd4). The HECT Nedd4 ∼Ub transitory intermediate provides a structural basis for the proposed sequential addition mechanism. The donor Ub, transferred from the E2, is bound to the Nedd4 C lobe with its C-terminal tail locked in an extended conformation, primed for catalysis. We provide evidence that the Nedd4-family members are Lys63-specific enzymes whose catalysis is mediated by an essential C-terminal acidic residue. © 2013 Nature America, Inc. All rights reserved.


Mari S.,Fondazione Instituto Firc Of Oncologia Molecolare | Ruetalo N.,Max Planck Institute for Developmental Biology | Maspero E.,Fondazione Instituto Firc Of Oncologia Molecolare | Stoffregen M.C.,Max Planck Institute for Developmental Biology | And 4 more authors.
Structure | Year: 2014

Nedd4-family ubiquitin ligases are key regulators of cell surface receptor signaling. Their dysregulation is associated with several human diseases, including cancer. Under normal conditions, the activity of various Nedd4 E3s is controlled through an autoinhibitory interaction of the N-terminal C2 domain with the C-terminal catalytic HECT domain. Here, we report the structural and functional framework for this intramolecular interaction. Our nuclear magnetic resonance (NMR) data and biochemical analyses on Smurf2 and Nedd4 show that the C2 domain has the potential to regulate E3 activity by maintaining the HECT domain in a low-activity state where its ability for transthiolation and noncovalent Ub binding are impaired. © 2014 Elsevier Ltd.


Pierotti M.A.,Instituto Nazionale Dei Tumori | Berrino F.,Instituto Nazionale Dei Tumori | Gariboldi M.,Fondazione IRCCS Instituto Nazionale Dei Tumori | Gariboldi M.,Fondazione Instituto Firc Of Oncologia Molecolare | And 6 more authors.
Oncogene | Year: 2013

Understanding the complexity of cancer and of the underlying regulatory networks provides a new paradigm that tackles cancer development and treatment through a system biology approach, contemporarily acting on various intersecting pathways. Cancer cell metabolism is an old pathogenetic issue that has recently gained new interest as target for therapeutic approaches. More than 70 years ago, Warburg discovered that malignant cells generally have altered metabolism with high rates of glucose uptake and increased glycolysis, even under aerobic condition. Observational studies have provided evidence that impaired metabolism, obesity, hyperglycemia and hyperinsulinemia may have a role in cancer development, progression and prognosis, and actually diabetic and obese patients have increased cancer risk. On the other hand, caloric restriction has been shown to prolong life span and reduce cancer incidence in several animal models, having an impact on different metabolic pathways. Metformin, an antidiabetic drug widely used for over 40 years, mimics caloric restriction acting on cell metabolism at multiple levels, reducing all energy-consuming processes in the cells, including cell proliferation. By overviewing molecular mechanisms of action, epidemiological evidences, experimental data in tumor models and early clinical study results, this review provides information supporting the promising use of metformin in cancer prevention and treatment. © 2013 Macmillan Publishers Limited All rights reserved.


Inui M.,University of Padua | Manfrin A.,University of Padua | Mamidi A.,University of Padua | Martello G.,University of Padua | And 8 more authors.
Nature Cell Biology | Year: 2011

The TGFβ pathway is critical for embryonic development and adult tissue homeostasis. On ligand stimulation, TGFβ and BMP receptors phosphorylate receptor-activated SMADs (R-SMADs), which then associate with SMAD4 to form a transcriptional complex that regulates gene expression through specific DNA recognition. Several ubiquitin ligases serve as inhibitors of R-SMADs, yet no deubiquitylating enzyme (DUB) for these molecules has so far been identified. This has left unexplored the possibility that ubiquitylation of R-SMADs is reversible and engaged in regulating SMAD function, in addition to degradation. Here we identify USP15 as a DUB for R-SMADs. USP15 is required for TGFβ and BMP responses in mammalian cells and Xenopus embryos. At the biochemical level, USP15 primarily opposes R-SMAD monoubiquitylation, which targets the DNA-binding domains of R-SMADs and prevents promoter recognition. As such, USP15 is critical for the occupancy of endogenous target promoters by the SMAD complex. These data identify an additional layer of control by which the ubiquitin system regulates TGFβ biology. © 2011 Macmillan Publishers Limited. All rights reserved.


Rangam G.,Fondazione Instituto Firc Of Oncologia Molecolare | Rangam G.,London Research Institute | Schmitz K.-M.,Fondazione Instituto Firc Of Oncologia Molecolare | Cobb A.J.A.,University of Reading | And 2 more authors.
PLoS ONE | Year: 2012

Activation induced deaminase (AID) deaminates cytosine to uracil, which is required for a functional humoral immune system. Previous work demonstrated, that AID also deaminates 5-methylcytosine (5 mC). Recently, a novel vertebrate modification (5-hydroxymethylcytosine - 5 hmC) has been implicated in functioning in epigenetic reprogramming, yet no molecular pathway explaining the removal of 5 hmC has been identified. AID has been suggested to deaminate 5 hmC, with the 5 hmU product being repaired by base excision repair pathways back to cytosine. Here we demonstrate that AID's enzymatic activity is inversely proportional to the electron cloud size of C5-cytosine - H > F > methyl >> hydroxymethyl. This makes AID an unlikely candidate to be part of 5 hmC removal. © 2012 Rangam et al.


Polo S.,Fondazione Instituto Firc Of Oncologia Molecolare | Polo S.,University of Milan
BMC Biology | Year: 2012

Ubiquitin-dependent regulation of endocytosis plays an important part in the control of signal transduction, and a critical issue in the understanding of signal transduction therefore relates to regulation of ubiquitination in the endocytic pathway. We discuss here what is known of the mechanisms by which signaling controls the activity of the ubiquitin ligases that specifically recognize the targets of ubiquitination on the endocytic pathway, and suggest alternative mechanisms that deserve experimental investigation. © 2012 Polo; licensee BioMed Central Ltd.


Radice P.,Unit of Molecular Bases of Genetic Risk and Genetic Testing | Radice P.,Fondazione Instituto Firc Of Oncologia Molecolare | de Summa S.,National Cancer Center Giovanni Paolo | Caleca L.,Unit of Molecular Bases of Genetic Risk and Genetic Testing | Tommasi S.,National Cancer Center Giovanni Paolo
Annals of Oncology | Year: 2011

In the last few years, several studies have focused on the interpretation of unclassified variants (UVs) of BRCA1 and BRCA2 genes. Analysis of UVs through a unique approach is not sufficient to understand their role in the development of tumors. Thus, it is clear that assembling results from different sources (genetic and epidemiological data, histopathological features, and in vitro and in silico analyses) represents a powerful way to classify such variants. Building reliable integrated models for UV classification requires the joining of many working groups to collaborative consortia, allowing data exchange and improvements of methods. This will lead to improvement in the predictivity of gene testing in BRCA1 and BRCA2 and, consequently, to an increase in the number of families that can be correctly classified as linked or unlinked to these genes, allowing more accurate genetic counseling and clinical management. © The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.


Bisi S.,Fondazione Instituto Firc Of Oncologia Molecolare | Disanza A.,Fondazione Instituto Firc Of Oncologia Molecolare | Malinverno C.,Fondazione Instituto Firc Of Oncologia Molecolare | Frittoli E.,Fondazione Instituto Firc Of Oncologia Molecolare | And 3 more authors.
Current Opinion in Cell Biology | Year: 2013

The multimolecular WAVE regulatory (WRC) and Arp2/3 complexes are primarily responsible to generate pushing forces at migratory leading edges by promoting branch elongation of actin filaments. The architectural complexity of these units betrays the necessity to impose a tight control on their activity. This is exerted through temporally coordinated and coincident signals which limit the intensity and duration of this activity. In addition, interactions of the WRC and Arp2/3 complexes with membrane binding and surprisingly membrane trafficking proteins is also emerging, revealing the existence of an 'endocytic wiring system' that spatially restrict branched actin elongation for the execution of polarized functions during cell migration. © 2013 Elsevier Ltd.


Pece S.,Fondazione Instituto Firc Of Oncologia Molecolare | Pece S.,University of Milan | Pece S.,Instituto Europeo Of Oncologia | Tosoni D.,Fondazione Instituto Firc Of Oncologia Molecolare | And 13 more authors.
Cell | Year: 2010

Pathways that govern stem cell (SC) function are often subverted in cancer. Here, we report the isolation to near purity of human normal mammary SCs (hNMSCs), from cultured mammospheres, on the basis of their ability to retain the lipophilic dye PKH26 as a consequence of their quiescent nature. PKH26-positive cells possess all the characteristics of hNMSCs. The transcriptional profile of PKH26-positive cells (hNMSC signature) was able to predict biological and molecular features of breast cancers. By using markers of the hNMSC signature, we prospectively isolated SCs from the normal gland and from breast tumors. Poorly differentiated (G3) cancers displayed higher content of prospectively isolated cancer SCs (CSCs) than did well-differentiated (G1) cancers. By comparing G3 and G1 tumors in xenotransplantation experiments, we directly demonstrated that G3s are enriched in CSCs. Our data support the notion that the heterogeneous phenotypical and molecular traits of human breast cancers are a function of their CSC content. © 2010 Elsevier Inc. All rights reserved.

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