title: Low expression of the E3 ubiquitin ligase CBL confers chemoresistance in human pancreatic cancer and is targeted by epidermal growth factor receptor inhibition.
abstract: OBJECTIVE
  Expression of CBL, an ubiquitin ligase, is decreased in 60% of human pancreatic ductal adenocarcinomas (PDAC) and is associated with shorter overall survival. We sought to determine how low CBL directly contributes to clinically more aggressive PDAC.
  
  
  METHODS
  Human PDACs were stained for CBL, pEGFR, and EGFR. CBL-low was modeled in PDAC cells (Panc-1, L3.6pl, and AsPC-1) via transient transfection (siRNA) or stable knockdown (shRNA). Cell viability and apoptosis were measured by MTT assays and FACS. Immunoblot and a phospho-receptor tyrosine kinase (pRTK) array were used to probe signal transduction. NOD-scid-IL2Rγ(null) mice were subcutaneously implanted with PDAC or PDAC(CBL-low) cells on opposite flanks and treated with gemcitabine ± erlotinib for ≥4 weeks.
  
  
  RESULTS
  There was an inverse correlation between CBL and pEGFR protein expression in 12 of 15 tumors. CBL knockdown increased PDAC resistance to gemcitabine and 5-fluorouracil (5-FU) by upregulating pEGFR (Y1068), pERK, and pAKT. A pRTK array of PDAC(CBL-low) cells revealed additional activated tyrosine kinases but all to a much lower magnitude than EGFR. Increased chemoresistance from low CBL was abrogated by the EGFR inhibitor erlotinib both in vitro and in vivo. Erlotinib+gemcitabine-treated PDAC(CBL-low) cells exhibited greater apoptosis by cleaved PARP, caspase-3, and Annexin V/PI.
  
  
  CONCLUSIONS
  Low CBL causes chemoresistance in PDAC via stress-induced EGFR activation that can be effectively abrogated by EGFR inhibition. These results suggest that dysregulation of ubiquitination is a key mechanism of EGFR hyperactivation in PDAC and that low CBL may define PDAC tumors likely to respond to erlotinib treatment.
authors.affiliation: Department of Surgery, Division of General Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
authors.forename: Brian E
authors.initials: BE
authors.lastname: Kadera
authors.affiliation: Department of Surgery, Division of General Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Toste
authors.affiliation: Department of Surgery, Division of General Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
authors.forename: Nanping
authors.initials: N
authors.lastname: Wu
authors.affiliation: Department of Surgery, Division of General Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
authors.forename: Luyi
authors.initials: L
authors.lastname: Li
authors.affiliation: Department of Surgery, Division of General Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Nguyen
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California. Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
authors.forename: David W
authors.initials: DW
authors.lastname: Dawson
authors.affiliation: Department of Surgery, Division of General Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California. Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California. Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California. tdonahue@mednet.ucla.edu.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Donahue
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D000818:Animals; D021441:Carcinoma, Pancreatic Ductal / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D053148:Caspase 3 / Q000096:biosynthesis; D045744:Cell Line, Tumor; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D000069347:Erlotinib Hydrochloride; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051379:Mice; D050721:Proto-Oncogene Proteins c-cbl / Q000096:biosynthesis* / Q000235:genetics; D011799:Quinazolines / Q000008:administration & dosage; D034741:RNA, Small Interfering; D000093542:Gemcitabine
chemical_list: D011799:Quinazolines; D034741:RNA, Small Interfering; D003841:Deoxycytidine; D000069347:Erlotinib Hydrochloride; D050721:Proto-Oncogene Proteins c-cbl; D066246:ErbB Receptors; D053148:Caspase 3; C067419:CBL protein, human; D000093542:Gemcitabine