title: Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer.
abstract: BACKGROUND
  The antibody cetuximab, targeting epidermal growth factor receptor (EGFR), is used to treat metastatic colorectal cancer (mCRC). Clinical trials suggest reduced benefit from the combination of cetuximab with oxaliplatin. The aim of this study was to investigate potential negative interactions between cetuximab and oxaliplatin.
  
  
  METHODS
  Thiazolyl blue tetrazolium bromide (MTT) assay and Calcusyn software were used to characterize drug interactions. Reactive oxygen species (ROS) were measured by flow cytometry and real-time polymerase chain reaction oxidative stress arrays identified genes regulating ROS production. Chromatin immunoprecipitation (ChIP) measured signal transducer and activator of transcription 1 (STAT-1) binding to dual oxidase 2 (DUOX2) promoter. SW48, DLD-1 KRAS wild-type cell lines and DLD-1 xenograft models exposed to cetuximab, oxaliplatin, or oxaliplatin + cetuximab (control [saline]; n = 3 mice per treatment group) were used. Statistical tests were two-sided.
  
  
  RESULTS
  Cetuximab and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, P = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, P < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity.
  
  
  CONCLUSIONS
  Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic agents in future therapeutic use.
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: Valeria
authors.initials: V
authors.lastname: Santoro
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: Ruochen
authors.initials: R
authors.lastname: Jia
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: Hannah
authors.initials: H
authors.lastname: Thompson
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: Anke
authors.initials: A
authors.lastname: Nijhuis
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: Rosemary
authors.initials: R
authors.lastname: Jeffery
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Kiakos
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Silver
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS);
authors.forename: John A
authors.initials: JA
authors.lastname: Hartley
authors.affiliation: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London, London, UK (VS, RJ, KK, JAH, DH); Colorectal Cancer Genetics Group, Blizard Institute, London, UK (HT, AN, RJ, ARS); d.hochhauser@ucl.ac.uk.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hochhauser
mesh_terms: D000818:Animals; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D000068818:Cetuximab / Q000494:pharmacology* / Q000627:therapeutic use; D047369:Chromatin Immunoprecipitation; D015179:Colorectal Neoplasms / Q000188:drug therapy*; D000074623:Dual Oxidases; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D064593:Heterografts; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019255:NADPH Oxidases / Q000378:metabolism; D009944:Organoplatinum Compounds / Q000037:antagonists & inhibitors* / Q000627:therapeutic use; D000077150:Oxaliplatin; D017382:Reactive Oxygen Species / Q000378:metabolism*; D050794:STAT1 Transcription Factor / Q000378:metabolism
chemical_list: D009944:Organoplatinum Compounds; D017382:Reactive Oxygen Species; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D000077150:Oxaliplatin; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431515:DUOX2 protein, human; C512478:EGFR protein, human; D066246:ErbB Receptors; D000068818:Cetuximab