Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

JNCI: Journal of the National Cancer Institute, Jun 2016

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.

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Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

JNCI J Natl Cancer Inst ( Cancer Valeria Santoro 0 1 2 Ruochen Jia 0 1 2 Hannah Thompson 0 1 2 Anke Nijhuis 0 1 2 Rosemary Jeffery 0 1 2 Konstantinos Kiakos 0 1 2 Andrew R. Silver 0 1 2 John A. Hartley 0 1 2 0 DH); Colorectal Cancer Genetics Group, Blizard Institute , London, UK, HT, AN, RJ, ARS 1 Affiliations of authors: Cancer Research UK Drug-DNA Interactions Research Group, UCL Cancer Institute, University College London , London, UK (VS, RJ, KK, JAH 2 15. McHugh PJ, Spanswick VJ, Hartley JA. Repair of DNA interstrand crosslinks: 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 wildtype 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 2.1P1 ,= .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 STAT1mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.P41<, .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. In view of the importance of the epidermal growth fa-c Abrogation; Colorectal - inhibitors (1–4). Antibodies inhibiting EGFR, including cetux-i tor receptor (EGFR) in the development and maintenance of mab and panitumumab, have shown efficacy in colorectal human cancers, there is considerable interest in inhibiting cancer (CRC) either as monotherapy, or in combination with this pathway with monoclonal antibodies or small molecule chemotherapy (5–8). 1 of 11 Preclinical and clinical studies of cetuximab or panitumumab Drug Combination Assays with irinotecan-based chemotherapy have shown benefit in CRC ( 9–10 ). In contrast, despite some efficacy for antibodies targeting Ten thousand cells per well were seeded in a 96-well plate EGFR and oxaliplatin combinations1( 1–12 ), other studies have suggested either no benefit or a negative interaction. A  ra-nd omized study using cetuximab in combination with oxaliplatin and fluoropyrimidines to treat CRC showed no benefit from addition of cetuximab 1(3). More recently, the randomized NEW (Corning) and drug-treated for 72 hours with cetuximab, ox-ali platin, SN-38, or their combination; inhibition of proliferation was measured by MTT assay. Synergy or antagonism were determined with Calcusyn software using methodology of Chou and Talalay 2(3). Drug scheduling and dosing is provided in the EPOC study of oxaliplatin and 5-fluorouracil alone or combined Supplementary Material(savailable online). with cetuximab demonstrated reduced progression-free and overall survival with cetuximab14(). cross-links, DNA-protein adducts1( 5–17 ), and generate forma Cisplatin and oxaliplatin induce intra- and interstrand DNA Stress Arrays Real-Time Polymerase Chain Reaction Oxidative tion of reactive oxygen species (ROS) and toxic oxygen meta-b Real-time polymerase chain reaction (RT-PCR) oxidative stress olites, which cause cytotoxic effects by inducing DNA damage arrays (Qiagen) were used to measure RNA expression of stressand apoptosis (18–21). Given lack of synergy and possible anta-g related genes following cetuximab and oxaliplatin treatment. onism of oxaliplatin combined with cetuximab in CRC, we Additional details are provided in thSeupplementary Materials investigated potential mechanisms of interaction. (available online). Methods Reagents and Antibodies Cetuximab (5mg/mL) was obtained from Merck Serono KGaA (Darmst (...truncated)


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Santoro, Valeria, Jia, Ruochen, Thompson, Hannah, Nijhuis, Anke, Jeffery, Rosemary, Kiakos, Konstantinos, Silver, Andrew R., Hartley, John A., Hochhauser, Daniel. Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer, JNCI: Journal of the National Cancer Institute, 2016, Volume 108, Issue 6, DOI: 10.1093/jnci/djv394