title: Impaired degradation followed by enhanced recycling of epidermal growth factor receptor caused by hypo-phosphorylation of tyrosine 1045 in RBE cells.
abstract: BACKGROUND
  Since cholangiocarcinoma has a poor prognosis, several epidermal growth factor receptor (EGFR)-targeted therapies with antibody or small molecule inhibitor treatment have been proposed. However, their effect remains limited. The present study sought to understand the molecular genetic characteristics of cholangiocarcinoma related to EGFR, with emphasis on its degradation and recycling.
  
  
  METHODS
  We evaluated EGFR expression and colocalization by immunoblotting and immunofluorescence, cell surface EGFR expression by fluorescence-activated cell sorting (FACS), and EGFR ubiquitination and protein binding by immunoprecipitation in the human cholangiocarcinoma RBE and immortalized cholangiocyte MMNK-1 cell lines. Monensin treatment and Rab11a depletion by siRNA were adopted for inhibition of EGFR recycling.
  
  
  RESULTS
  Upon stimulation with EGF, ligand-induced EGFR degradation was impaired and the expression of phospho-tyrosine 1068 and phospho-p44/42 MAPK was sustained in RBE cells as compared with MMNK-1 cells. In RBE cells, the process of EGFR sorting for lysosomal degradation was blocked at the early endosome stage, and non-degradated EGFR was recycled to the cell surface. A disrupted association between EGFR and the E3 ubiquitin ligase c-Cbl, as well as hypo-phosphorylation of EGFR at tyrosine 1045 (Tyr1045), were also observed in RBE cells.
  
  
  CONCLUSIONS
  In RBE cells, up-regulation of EGFR Tyr1045 phosphorylation is a potentially useful molecular alteration in EGFR-targeted therapy. The combination of molecular-targeted therapy determined by the characteristics of individual EGFR phosphorylation events and EGFR recycling inhibition show promise in future treatments of cholangiocarcinoma.
authors.affiliation: First Department of Surgery, Shinshu University School of Medicine, Asahi, Matsumoto, Nagano, Japan.
authors.forename: Anping
authors.initials: A
authors.lastname: Gui
authors.forename: Akira
authors.initials: A
authors.lastname: Kobayashi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Motoyama
authors.forename: Masato
authors.initials: M
authors.lastname: Kitazawa
authors.forename: Michiko
authors.initials: M
authors.lastname: Takeoka
authors.forename: Shinichi
authors.initials: S
authors.lastname: Miyagawa
mesh_terms: D001650:Bile Duct Neoplasms / Q000235:genetics / Q000378:metabolism*; D001653:Bile Ducts, Intrahepatic; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D021381:Protein Transport; D059748:Proteolysis / Q000187:drug effects; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014443:Tyrosine / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D014443:Tyrosine; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D066246:ErbB Receptors