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Saviore dell'Adamello, Italy

Dey J.H.,Friedrich Miescher Institute for Biomedical Research | Bianchi F.,IFOM | 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. Source

Du S.,University of Toronto | Draper B.W.,University of California at Davis | Mione M.,IFOM | Moens C.B.,Fred Hutchinson Cancer Research Center | Bruce A.,University of Toronto
Developmental Biology | Year: 2012

The T-box transcription factor Eomesodermin (Eomes) has been implicated in patterning and morphogenesis in frog, fish and mouse. In zebrafish, one of the two Eomes homologs, Eomesa, has been implicated in dorsal-ventral patterning, epiboly and endoderm specification in experiments employing over-expression, dominant-negative constructs and antisense morpholino oligonucleotides. Here we report for the first time the identification and characterization of an Eomesa mutant generated by TILLING. We find that Eomesa has a strictly maternal role in the initiation of epiboly, which involves doming of the yolk cell up into the overlying blastoderm. By contrast, epiboly progression is normal, demonstrating for the first time that epiboly initiation is genetically separable from progression. The yolk cell microtubules, which are required for epiboly, are defective in maternal-zygotic eomesa mutant embryos. In addition, the deep cells of the blastoderm are more tightly packed and exhibit more bleb-like protrusions than cells in control embryos. We postulate that the doming delay may be the consequence both of overly stabilized yolk cell microtubules and defects in the adhesive properties or motility of deep cells. We also show that Eomesa is required for normal expression of the endoderm markers sox32, bon and og9x; however it is not essential for endoderm formation. © 2011 Elsevier Inc. Source

Ray Chaudhuri A.,University of Zurich | Hashimoto Y.,London Research Institute | Hashimoto Y.,University of Salamanca | Herrador R.,University of Zurich | And 9 more authors.
Nature Structural and Molecular Biology | Year: 2012

Topoisomerase I (Top1) releases torsional stress during DNA replication and transcription and is inhibited by camptothecin and camptothecin-derived cancer chemotherapeutics. Top1 inhibitor cytotoxicity is frequently linked to double-strand break (DSB) formation as a result of Top1 being trapped on a nicked DNA intermediate in replicating cells. Here we use yeast, mammalian cell lines and Xenopus laevis egg extracts to show that Top1 poisons rapidly induce replication-fork slowing and reversal, which can be uncoupled from DSB formation at sublethal inhibitor doses. Poly(ADP-ribose) polymerase activity, but not single-stranded break repair in general, is required for effective fork reversal and limits DSB formation. These data identify fork reversal as a means to prevent chromosome breakage upon exogenous replication stress and implicate proteins involved in fork reversal or restart as factors modulating the cytotoxicity of replication stress-inducing chemotherapeutics. © 2012 Nature America, Inc. All rights reserved. Source

Polo S.,IFOM | 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. Source

Mari S.,IFOM | Ruetalo N.,Max Planck Institute for Developmental Biology | Maspero E.,IFOM | 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. Source

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