title: ERK phosphorylation predicts synergism between gemcitabine and the epidermal growth factor receptor inhibitor AG1478.
abstract: BACKGROUND
  Clinical trials combining epidermal growth factor receptor (EGFR) inhibitors with gemcitabine-based chemotherapy in non-small cell lung cancer (NSCLC) have not produced a survival advantage. This may be caused by antagonism between the two drugs or mutations that promote such, possibly RAS mutation. Furthermore, ERK, a critical growth regulator downstream of RAS, may play a role. This study aimed to explore the relationship between ERK, synergy/antagonism and cell cycle arrest in combination treatment.
  
  
  METHODS
  A549 (mutant KRAS), H322 (wildtype KRAS) and siRNA-mediated KRAS knockdown A549 were treated with gemcitabine and/or the EGFR inhibitor AG1478 and analyzed with median effect analysis. Cell cycle distribution and ERK phosphorylation were assessed using flow cytometry and ELISA, respectively. Effect on cytotoxicity after ERK inhibition by U0126 was also assessed.
  
  
  RESULTS
  Cytotoxic interaction was dose dependent with antagonism at high dose AG1478. G1 arrest was observed with both high dose AG1478 and high dose gemcitabine and therefore was inconsistently associated with antagonism. Furthermore, ERK phosphorylation was increased by gemcitabine and its suppression by AG1478 was related to antagonism particularly in H322. ERK's effect in antagonism was further confirmed by using U0126. Greater antagonism was observed in the KRAS mutant cell line and KRAS knockdown by siRNA resulted in increased sensitivity to AG1478 as well as combination treatment.
  
  
  CONCLUSIONS
  Our findings are consistent with a model in which ERK phosphorylation favors synergy and the outcome depends on the balance between gemcitabine-induced and AG1478-inhibited ERK phosphorylation. KRAS mutation confers resistance to AG1478 as well as combination treatment.
authors.affiliation: Cancer Pharmacology and Therapeutic Laboratory, Medical Oncology, St. George Hospital, Sydney, NSW, Australia. Peter.Luk@sesiahs.health.nsw.gov.au
authors.forename: Peter P
authors.initials: PP
authors.lastname: Luk
authors.forename: Peter
authors.initials: P
authors.lastname: Galettis
authors.forename: Matthew
authors.initials: M
authors.lastname: Links
mesh_terms: D000230:Adenocarcinoma / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D002070:Butadienes / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D003841:Deoxycytidine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004357:Drug Synergism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D006801:Humans; D009154:Mutation / Q000235:genetics; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D011799:Quinazolines; D034741:RNA, Small Interfering / Q000235:genetics; D020032:Tyrphostins / Q000008:administration & dosage / Q000494:pharmacology*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*; D000093542:Gemcitabine
chemical_list: D002070:Butadienes; C117307:KRAS protein, human; D009570:Nitriles; D011518:Proto-Oncogene Proteins; D011799:Quinazolines; D034741:RNA, Small Interfering; D020032:Tyrphostins; C113580:U 0126; D003841:Deoxycytidine; C101044:RTKI cpd; D066246:ErbB Receptors; D048049:Extracellular Signal-Regulated MAP Kinases; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D000093542:Gemcitabine