title: A nanobody targeting the F-actin capping protein CapG restrains breast cancer metastasis.
abstract: BACKGROUND
  Aberrant turnover of the actin cytoskeleton is intimately associated with cancer cell migration and invasion. Frequently however, evidence is circumstantial, and a reliable assessment of the therapeutic significance of a gene product is offset by lack of inhibitors that target biologic properties of a protein, as most conventional drugs do, instead of the corresponding gene. Proteomic studies have demonstrated overexpression of CapG, a constituent of the actin cytoskeleton, in breast cancer. Indirect evidence suggests that CapG is involved in tumor cell dissemination and metastasis. In this study, we used llama-derived CapG single-domain antibodies or nanobodies in a breast cancer metastasis model to address whether inhibition of CapG activity holds therapeutic merit.
  
  
  METHODS
  We raised single-domain antibodies (nanobodies) against human CapG and used these as intrabodies (immunomodulation) after lentiviral transduction of breast cancer cells. Functional characterization of nanobodies was performed to identify which biochemical properties of CapG are perturbed. Orthotopic and tail vein in vivo models of metastasis in nude mice were used to assess cancer cell spreading.
  
  
  RESULTS
  With G-actin and F-actin binding assays, we identified a CapG nanobody that binds with nanomolar affinity to the first CapG domain. Consequently, CapG interaction with actin monomers or actin filaments is blocked. Intracellular delocalization experiments demonstrated that the nanobody interacts with CapG in the cytoplasmic environment. Expression of the nanobody in breast cancer cells restrained cell migration and Matrigel invasion. Notably, the nanobody prevented formation of lung metastatic lesions in orthotopic xenograft and tail-vein models of metastasis in immunodeficient mice. We showed that CapG nanobodies can be delivered into cancer cells by using bacteria harboring a type III protein secretion system (T3SS).
  
  
  CONCLUSIONS
  CapG inhibition strongly reduces breast cancer metastasis. A nanobody-based approach offers a fast track for gauging the therapeutic merit of drug targets. Mapping of the nanobody-CapG interface may provide a platform for rational design of pharmacologic compounds.
authors: Katrien
authors: K
authors: Van Impe
authors: Jonas
authors: J
authors: Bethuyne
authors: Steven
authors: S
authors: Cool
authors: Francis
authors: F
authors: Impens
authors: David
authors: D
authors: Ruano-Gallego
authors: Olivier
authors: O
authors: De Wever
authors: Berlinda
authors: B
authors: Vanloo
authors: Marleen
authors: M
authors: Van Troys
authors: Kathleen
authors: K
authors: Lambein
authors: Ciska
authors: C
authors: Boucherie
authors: Evelien
authors: E
authors: Martens
authors: Olivier
authors: O
authors: Zwaenepoel
authors: Gholamreza
authors: G
authors: Hassanzadeh-Ghassabeh
authors: Joël
authors: J
authors: Vandekerckhove
authors: Kris
authors: K
authors: Gevaert
authors: Luis Ángel
authors: LÁ
authors: Fernández
authors: Niek N
authors: NN
authors: Sanders
authors: Jan
authors: J
authors: Gettemans
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D016513:Mice, SCID; D008840:Microfilament Proteins / Q000235:genetics / Q000276:immunology*; D058990:Molecular Targeted Therapy / Q000379:methods*; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology*; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000494:pharmacology*
chemical_list: D000199:Actins; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D061905:Single-Domain Antibodies; C066575:CAPG protein, human