Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

et al. (2013) Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells. PLoS ONE 8(6): e66302. doi:10.1371/journal.pone.0066302 Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells Kohei Shigeta 0 Tetsu Hayashida 0 Yoshinori Hoshino 0 Koji Okabayashi 0 Takashi Endo 0 Yoshiyuki Ishii 0 Hirotoshi Hasegawa 0 Yuko Kitagawa 0 Anthony W.I. Lo, The Chinese University of Hong Kong, Hong Kong 0 Keio University, School of Medicine, Department of Surgery , Shinjuku-ku, Tokyo , Japan Cetuximab is a chimeric mouse-human monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). However, EGFR expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer (CRC) patients treated with cetuximab. Therefore, we evaluated the correlation between EGFR levels detected by cetuximab and drug sensitivities of CRC cell lines (Caco-2, WiDR, SW480, and HCT116) and the A431 epidermoid carcinoma cell line. We used flow cytometry (FCM) to detect EGFR-binding of biotinylated cetuximab on the cell surface. Subcloned cell lines showing the highest and lowest EGFR expression levels were chosen for further study. Cytotoxic assays were used to determine differential responses to cetuximab. Xenograft models treated with cetuximab intraperitoneally to assess sensitivity to cetuximab. Strong responses to cetuximab were specifically exhibited by subcloned cells with high EGFR expression levels. Furthermore, cetuximab inhibited the growth of tumors in xenograft models with high or low EGFR expression levels by 35% and 10%-20%, respectively. We conclude that detection of EGFR expression by cetuximab promises to provide a novel, sensitive, and specific method for predicting the sensitivity of CRC to cetuximab. - Funding: This research was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific Research (B) (#23791559 K.S., #23791499 T.H. and #25293292 Y.K.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The epidermal growth factor receptor (EGFR) is a member of the human EGFR family of receptor protein tyrosine kinases. It is an important therapeutic target in metastatic colorectal cancer (mCRC), and increased EGFR expression is the hallmark of many human tumors [1,2]. Activation of the EGFR signaling pathway results in increased tumor proliferation, angiogenesis, metastasis, and tumor invasiveness through the binding of a number of different ligands, including EGF-like molecules, transforming growth factor-a (TGFa), and neuregulins to the receptors ectodomain [3]. EGFR activation results in the initiation of potentially oncogenic intracellular signaling cascades, including the RAS-mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/Akt, phospholipase C, signal transducer and activator of transcription (STAT), and SRC/FAK pathways [46]. The development of monoclonal antibodies has improved the strategies for inhibiting the activity of the EGFR inhibition in cancer therapy. Cetuximab (Erbitux, Merck-Serono, Darmstadt, Germany) is a chimeric monoclonal antibody (IgG1) that binds to the ectodomain of the human EGFR and competitively inhibits ligand binding to suppress tumor proliferation. The efficacy of cetuximab was evaluated in combination with irinotecan to treat mCRC patients whose tumors are positive for EGFR expression (assessed by immunohistochemistry) and are resistant to FOLFOX or FOLFIRI regimens [710]. Many studies have been conducted to identify factors that can predict the response to treatment, and CRC with mutated KRAS was identified as a rule does not respond to anti-EGFR therapy. [11,12]. In contrast, factors such as EGFR over-expression, amplification of its gene, and p53 mutations correlate with the response to cetuximab; however, they are not completely effective in predicting the response to cetuximab therapy [13,14]. Experimental studies have suggested a correlation between the EGFR expression level and the efficacy of cetuximab [15]. However, this correlation seems to be speculative in the clinical setting, and some studies report that no relationship has been found between the intensity of the immunohistochemical staining for EGFR and the response rate [assessment was performed using Response Evaluation Criteria in Solid Tumors (RECIST)], progressive free survival, or overall survival in clinical trials [8,1618]. A recent report demonstrated that a mutation within the EGFR ectodomain confers resistance to cetuximab by preventing its binding [19]. Therefore, we speculated that the detection of EGFR using immunohistochemical staining using non-specific IgG1 antibody differs from detection by cetuximab. We further hypothesized that the cell membrane-specific EGFR expression levels, which can be detected by cetuximab, may influence the inhibition of cell proliferation. In this study, we devised a method, in which we used biotinylated cetuximab as the primary antibody for flow cytometry (FCM) to directly detect the EGFR expression by CRC cell lines. Using this technique we evaluated the relationship between EGFR levels detected by cetuximab-sensitivities of CRC cell lines. Materials and Methods Preparation of Biotinylated Cetuximab The mechanism by which biotinylated cetuximab binds to EGFR is shown in Figure 1a. Biotin was conjugated to cetuximab using an adaptation of the method described by Medical & Biological Laboratories Co., Ltd. Colon Cancer Cell Lines and Identification of EGFR Ectodomain Mutations, KRAS, BRAF, and PIK3CA Four human CRC cell lines, Caco-2, WiDR, SW480, HCT116, and epidermoid carcinoma cell line A431 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured as recommended. Authentication of all cell lines was performed by investigating the mutation status of each CRC cell line using the Scorpion-arms or direct sequence methods (conducted by SRS Co., Japan). KRAS (codons 12 and 13), BRAF (exon 15), PIK3CA (exons 9 and 20), and EGFR ectodomain (S492) mutations were determined in a subset of cell lines and results are summarized in Table 1. Caco-2, WiDR, SW480, and A431 were grown in Dulbeccos Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 5% 0.1 mM penicillinstreptomycin, and HCT116 was grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% FBS and 5% 0.1 mM penicillin streptomycin. All cell lines were incubated at 37uC in a humidified atmosphere containing 5% CO2. EGFR-binding of Biotinylated Cetuximab and Evaluation of EGFR Expression Levels u (...truncated)