title: ErbB-2 blockade and prenyltransferase inhibition alter epidermal growth factor and epidermal growth factor receptor trafficking and enhance (111)In-DTPA-hEGF Auger electron radiation therapy.
abstract: The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of (111)In-labeled human epidermal growth factor ((111)In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of (111)In-DTPA-hEGF.
  
  
  METHODS
  The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. (111)In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of (111)In-DTPA-hEGF in MDA-MB-468 (1.3 × 10(6) EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 × 10(6) EGFR per cell; 0.4 × 10(5) ErbB-2 per cell) cell lines. The cytotoxicity of (111)In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 μg/mL) or L-778,123 (0-22.5 μM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with (111)In-DTPA-hEGF plus trastuzumab or L-788,123.
  
  
  RESULTS
  Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of (111)In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to (111)In-DTPA-hEGF. Nuclear localization and cytotoxicity of (111)In-DTPA-hEGF were significantly increased in 231-H2N xenografts by cotreatment with L-788,123 (P < 0.0001).
  
  
  CONCLUSIONS
  The therapeutic efficacy of (111)In-DTPA-hEGF is increased through the coadministration of selected molecularly targeted drugs that modulate EGFR signaling and trafficking.
authors.affiliation: Department of Oncology, CRUK/MRC Gray Institute for Radiation Oncology and Biology, University of Oxford, Oxford, United Kingdom.
authors.forename: Bart
authors.initials: B
authors.lastname: Cornelissen
authors.forename: Sonali
authors.initials: S
authors.lastname: Darbar
authors.forename: Rebecca
authors.initials: R
authors.lastname: Hernandez
authors.forename: Veerle
authors.initials: V
authors.lastname: Kersemans
authors.forename: Iain
authors.initials: I
authors.lastname: Tullis
authors.forename: Paul R
authors.initials: PR
authors.lastname: Barber
authors.forename: Sean
authors.initials: S
authors.lastname: Smart
authors.forename: Borivoj
authors.initials: B
authors.lastname: Vojnovic
authors.forename: Raymond
authors.initials: R
authors.lastname: Reilly
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Vallis
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D003114:Colony-Forming Units Assay; D004122:Dimethylallyltranstransferase / Q000037:antagonists & inhibitors*; D004583:Electrons / Q000627:therapeutic use*; D004815:Epidermal Growth Factor / Q000378:metabolism* / Q000493:pharmacokinetics; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D021381:Protein Transport / Q000187:drug effects; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors*; D015398:Signal Transduction / Q000187:drug effects; D000068878:Trastuzumab
chemical_list: D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D004122:Dimethylallyltranstransferase; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D007093:Imidazoles; D004369:Pentetic Acid; D047428:Protein Kinase Inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C478252:111In-DTPA-human epidermal growth factor; C425231:L 778,123; C508053:ERBB2 protein, human