title: Single Chain Antibodies as Tools to Study transforming growth factor-β-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.
abstract: The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-β signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-β signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-β signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors of the insulin receptor, which decreased levels of p179SMAD3/SMAD4 complexes, thereby demonstrating the suitability of the recombinant affinity reagents applied in isPLA in screening for inhibitors of cell signaling.
authors.affiliation: From the ‡Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 85, Sweden; **YUMAB GmbH, Rebenring 33 Braunschweig 38106, Germany.
authors.forename: Andries
authors.initials: A
authors.lastname: Blokzijl
authors.affiliation: From the ‡Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 85, Sweden; agata.zieba@igp.uu.se.
authors.forename: Agata
authors.initials: A
authors.lastname: Zieba
authors.affiliation: ‖Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Department of Biotechnology, Spielmannstr. 7, Braunschweig 38106, Germany;
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.affiliation: ‖Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Department of Biotechnology, Spielmannstr. 7, Braunschweig 38106, Germany; **YUMAB GmbH, Rebenring 33 Braunschweig 38106, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schirrmann
authors.affiliation: ‖Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Department of Biotechnology, Spielmannstr. 7, Braunschweig 38106, Germany;
authors.forename: Saskia
authors.initials: S
authors.lastname: Helmsing
authors.affiliation: From the ‡Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 85, Sweden;
authors.forename: Karin
authors.initials: K
authors.lastname: Grannas
authors.affiliation: From the ‡Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 85, Sweden;
authors.forename: Ellen
authors.initials: E
authors.lastname: Hertz
authors.affiliation: §Ludwig Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala SE-751 24, Sweden.
authors.forename: Anita
authors.initials: A
authors.lastname: Moren
authors.affiliation: From the ‡Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 85, Sweden;
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: From the ‡Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 85, Sweden;
authors.forename: Ola
authors.initials: O
authors.lastname: Söderberg
authors.affiliation: §Ludwig Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala SE-751 24, Sweden, ¶Dept. of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 23, Sweden;
authors.forename: Aristidis
authors.initials: A
authors.lastname: Moustakas
authors.affiliation: ‖Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics, Department of Biotechnology, Spielmannstr. 7, Braunschweig 38106, Germany;
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.affiliation: From the ‡Dept. of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala SE-751 85, Sweden;
authors.forename: Ulf
authors.initials: U
authors.lastname: Landegren
mesh_terms: D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D006801:Humans; D061986:MCF-7 Cells; D019151:Peptide Library; D010766:Phosphorylation; D015398:Signal Transduction / Q000187:drug effects; D057127:Single-Chain Antibodies / Q000032:analysis*; D051785:Smad Proteins / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D019151:Peptide Library; D057127:Single-Chain Antibodies; D051785:Smad Proteins; D016212:Transforming Growth Factor beta