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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2019, Vol. 13 Issue (1) : 83-93
Growth suppression of colorectal cancer expressing S492R EGFR by monoclonal antibody CH12
Qiongna Dong1,2, Bizhi Shi1, Min Zhou1, Huiping Gao1, Xiaoying Luo1, Zonghai Li1, Hua Jiang1()
1. State Key Laboratory of Oncogenes & Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China
2. Department of Otolaryngology, South Campus, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China
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Colorectal cancer (CRC) is a common malignant tumor in the digestive tract, and 30%–85% of CRCs express epidermal growth factor receptors (EGFRs). Recently, treatments using cetuximab, also named C225, an anti-EGFR monoclonal antibody, for CRC have been demonstrated to cause an S492R mutation in EGFR. However, little is known about the biological function of S492R EGFR. Therefore, we attempted to elucidate its biological function in CRC cells and explore new treatment strategies for this mutant form. Our study indicated that EGFR and S492R EGFR accelerate the growth of CRC cells in vitro and in vivo and monoclonal antibody CH12, which specifically recognizes an EGFR tumor-specific epitope, can bind efficiently to S492R EGFR. Furthermore, mAb CH12 showed significantly stronger growth suppression activities and induced a more potent antibody-dependent cellular cytotoxicity effect on CRC cells bearing S492R EGFR than mAb C225. mAb CH12 obviously suppressed the growth of CRC xenografts with S492R EGFR mutations in vivo. Thus, mAb CH12 may be a promising therapeutic agent in treating patients with CRC bearing an S492R EGFR mutation.

Keywords S492R EGFR ectodomain mutation      colorectal cancer      mAb CH12      immunnotherapy     
Corresponding Authors: Hua Jiang   
Just Accepted Date: 28 December 2018   Online First Date: 22 January 2019    Issue Date: 12 March 2019
 Cite this article:   
Qiongna Dong,Bizhi Shi,Min Zhou, et al. Growth suppression of colorectal cancer expressing S492R EGFR by monoclonal antibody CH12[J]. Front. Med., 2019, 13(1): 83-93.
Fig.1  S492R EGFR expression in the established cell lines and S492R EGFR promoted tumorigenicity in colorectal carcinoma. (A and B) Cell extracts from S492R EGFR-transfected cells were subjected to Western blot analysis. The blot was incubated with an antibody against EGFR detected full-length EGFR (approximately 170 kDa) by using monoclonal antibody 7F4; GAPDH was used as a loading control. (C and D) In vitro cell growth of control and transfected colorectal carcinoma cell lines stably expressing EGFR and S492R EGFR by the CCK-8 Assay Kit. (E) S492R EGFR promoting the proliferation of colorectal carcinoma cell in vivo. HT-29, HT-29-EGFR, and HT-29-S492R EGFR cells (5×105) were injected into 4–6-week-old female BALB/c nude mice (n = 6), respectively. Six days later, the tumor volume was measured every other day. Data are expressed as mean tumor volume±SD.
Fig.2  FACS analysis of parental and transfected colorectal carcinoma cell lines stably expressing S492R EGFR. Cells were incubated with C225 (black line) and CH12 (green line) followed by FITC-conjugated goat anti-human IgG antibody. The negative control (PBS) fluorescence is plotted on each panel (red line).
Fig.3  In vitro growth suppression effects of cetuximab (C225) or CH12 on parental or S492R-EGFR-transfected human colorectal carcinoma cell lines. (A, B, D, E) Each cell line was treated with cetuximab or CH12 at concentration ranging from 20, 40, 80, 160 mg/mL for 72 h. Data are expressed as the percentage inhibition of cell growth±SD. (C and F) In vitro cell proliferation assay after 80 mg/mL of antibody therapy in S492R EGFR over-expressed colorectal carcinoma cell lines. Results were presented as three independent experiments.
Fig.4  Antibody-dependent cellular cytotoxicity mediated by mAb CH12 or C225 on HT-29, HT-29-S492R EGFR, Caco-2, and Caco-2-S492R EGFR cells. C225 or CH12-mediated ADCC activity with PBMCs from healthy donors at an effector: target cell ratio of 20:1. Antibody concentrations ranged from 1×10–3 to 1×101 mg/mL. Rituximab was used as an antibody control. Data are presented as mean percentage±SD of cytotoxicity of triplicate determinations. Results are representative of three separate experiments.
Fig.5  Antitumor effects of C225 and CH12 on colon cancer xenografts. HT-29, HT-29- EGFR, and HT-29-S492R EGFR cells (1×106) were subcutaneously injected into 4–6-week-old female BALB/c nude mice. When the tumors had reached a mean tumor volume of 100 mm3, the mice were randomly divided into three groups and treated with PBS, C225, or CH12. (A, E, I) Tumor growth curves. (B, F, J) Average weight of the isolated tumor tissues in each group. (C, G, K) Data are expressed as the percentage inhibition of tumor growth (P<0.05). (D, H, L) Photos of tumor body of three groups treated with PBS, C225, or CH12.
Fig.6  Mechanisms of antitumor activity following treatment with C225 and CH12. Established HT-29-S492R EGFR xenografts treated with PBS, C225, or CH12 as single agents were excised and prepared by homogenization in cell lysis buffer. Tumor lysates (40 mg) were then subjected to SDS-PAGE and immunoblotted for total EGFR, p-EGFR (Tyr1068), total ERK, p-ERK, total Akt, p-Akt (Thr308), p-Akt (Ser473), total STAT3, p-STAT3, cyclin D1, Bcl-2, as indicated. GAPDH was used as a loading control.
Fig.7  CH12 reduced proliferation and induced apoptosis in the HT-29-S492R EGFR tumor xenografts. (A) CH12 treatment led to less growth compared with other treatments in HT-29-S492R EGFR xenograft. Tumor sections were stained for Ki-67. The cell proliferative index was assessed as the percentage of total cells that were Ki-67 positive from six randomly selected high power fields (200×) in xenografts from six mice of each group. (B) Quantitative analysis of Ki-67 staining. (C) CH12 treatment led to an increase in apoptosis compared with control in HT-29-S492R EGFR xenografts. Apoptotic cells were detected using the TUNEL assay. The apoptotic index was assessed by the ratio of TUNEL-positive cells: total number of cells from six randomly selected high power fields (200×) in xenografts from six mice of each group. (D) Quantitative analysis of TUNEL assay. Data are presented as the mean±SE. *P<0.05 versus control group.
1 JCEncarnação, ASPires, RAAmaral, TJGonçalves, MLaranjo, JECasalta-Lopes, ACGonçalves, ABSarmento-Ribeiro, AMAbrantes, MFBotelho. Butyrate, a dietary fiber derivative that improves irinotecan effect in colon cancer cells. J Nutr Biochem 2018; 56: 183–192 pmid: 29587241
2 GGoel. Evolution of regorafenib from bench to bedside in colorectal cancer: is it an attractive option or merely a “me too” drug? Cancer Manag Res 2018; 10: 425–437 pmid: 29563833
3 JHStrickler, HI Hurwitz. Palliative treatment of metastatic colorectal cancer: what is the optimal approach? Curr Oncol Rep 2014; 16(1): 363 pmid: 24293074
4 AKalyan, S Kircher, HShah, MMulcahy, ABenson. Updates on immunotherapy for colorectal cancer. J Gastrointest Oncol 2018; 9(1): 160–169 pmid: 29564182
5 MHDietvorst, FA Eskens. Current and novel treatment options for metastatic colorectal cancer: emphasis on aflibercept. Biol Ther 2013; 3(1): 25–33 pmid: 24392302
6 ESanz-Garcia, J Grasselli, GArgiles, MEElez, JTabernero. Current and advancing treatments for metastatic colorectal cancer. Expert Opin Biol Ther 2016; 16(1): 93–110 pmid: 26549053
7 SYLee, SC Oh. Advances of targeted therapy in treatment of unresectable metastatic colorectal cancer. BioMed Res Int 2016; 2016: 7590245 pmid: 27127793
8 CTomida, K Aibara, NYamagishi, CYano, H Nagano, TAbe, AOhno, K Hirasaka, TNikawa, STeshima-Kondo. The malignant progression effects of regorafenib in human colon cancer cells. J Med Invest 2015; 62(3-4): 195–198 pmid: 26399347
9 LLiang, L Wang, PZhu, YXia, Y Qiao, JWu, WZhuang, JFei, Y Wen, XJiang. A pilot study of apatinib as third-line treatment in patients with heavily treated metastatic colorectal cancer. Clin Colorectal Cancer 2018; 17(3): e443–e449 pmid: 29576426
10 CTomida, H Nagano, NYamagishi, TUchida, AOhno, K Hirasaka, TNikawa, STeshima-Kondo. Regorafenib induces adaptive resistance of colorectal cancer cells via inhibition of vascular endothelial growth factor receptor. J Med Invest 2017; 64 (3.4): 262–265 10.2152/jmi.64.262
11 TYamaoka, M Ohba, TOhmori. Molecular-targeted therapies for epidermal growth factor receptor and its resistance mechanisms. Int J Mol Sci 2017; 18(11): E2420 pmid: 29140271
12 PSavage, A Blanchet-Cohen, TRevil, DBadescu, SMISaleh, YCWang, DZuo, L Liu, NRBertos, VMunoz-Ramos, MBasik, KPetrecca, JAsselah, SMeterissian, MCGuiot, AOmeroglu, CLKleinman, MPark, J Ragoussis. A targetable EGFR-dependent tumor-initiating program in breast cancer. Cell Reports 2017; 21(5): 1140–1149 pmid: 29091754
13 PZhou, J Hu, XWang, JWang, Y Zhang, CWang. Epidermal growth factor receptor expression affects proliferation and apoptosis in non-small cell lung cancer cells via the extracellular signal-regulated kinase/microRNA 200a signaling pathway. Oncol Lett 2018; 15(4): 5201–5207 pmid: 29552158
14 JLi, R Liang, CSong, YXiang, YLiu. Prognostic significance of epidermal growth factor receptor expression in glioma patients. OncoTargets Ther 2018; 11: 731–742 pmid: 29445288
15 QLiu, S Yu, WZhao, SQin, Q Chu, KWu. EGFR-TKIs resistance via EGFR-independent signaling pathways. Mol Cancer 2018; 17(1): 53 pmid: 29455669
16 VTBroadbridge, CS Karapetis, TJPrice. Cetuximab in metastatic colorectal cancer. Expert Rev Anticancer Ther 2012; 12(5): 555–565 pmid: 22594891
17 MDel Prete, R Giampieri, LFaloppi, MBianconi, ABittoni, KAndrikou, SCascinu. Panitumumab for the treatment of metastatic colorectal cancer: a review. Immunotherapy 2015; 7(7): 721–738 pmid: 26250414
18 CMontagut, A Dalmases, BBellosillo, MCrespo, SPairet, MIglesias, MSalido, MGallen, SMarsters, SPTsai, AMinoche, SSeshagiri, SSerrano, HHimmelbauer, JBellmunt, ARovira, JSettleman, FBosch, JAlbanell. Identification of a mutation in the extracellular domain of the epidermal growth factor receptor conferring cetuximab resistance in colorectal cancer. Nat Med 2012; 18(2): 221–223 pmid: 22270724
19 CEsposito, AM Rachiglio, MLLa Porta, ASacco, CRoma, A Iannaccone, FTatangelo, LForgione, RPasquale, ABarbaro, GBotti, FCiardiello, NNormanno. The S492R EGFR ectodomain mutation is never detected in KRAS wild-type colorectal carcinoma before exposure to EGFR monoclonal antibodies. Cancer Biol Ther 2013; 14(12): 1143–1146 pmid: 24025416
20 DTougeron, U Cortes, AFerru, CVillalva, CSilvain, JMTourani, PLevillain, LKarayan-Tapon. Epidermal growth factor receptor (EGFR) and KRAS mutations during chemotherapy plus anti-EGFR monoclonal antibody treatment in metastatic colorectal cancer. Cancer Chemother Pharmacol 2013; 72(2): 397–403 pmid: 23765179
21 KNewhall, T Price, MPeeters, TWKim, J Li, SCascinu, PRuff, AS Suresh, AThomas, STjulandin, SOgbagabriel, MBoedigheimer, SSexson, KZhang, SMurugappan, RSidhu. Frequency of S492R mutations in the epidermal growth factor receptor: analysis of plasma DNA from metastatic colorectal cancer patients treated with panitumumab or cetuximab monotherapy. Ann Oncol 2014; 25 (suppl_2): ii109
22 HJiang, H Wang, ZTan, SHu, H Wang, BShi, LYang, P Li, JGu, HWang, Z Li. Growth suppression of human hepatocellular carcinoma xenografts by a monoclonal antibody CH12 directed to epidermal growth factor receptor variant III. J Biol Chem 2011; 286(7): 5913–5920 pmid: 21163950
23 HWang, B Shi, QZhang, HJiang, SHu, J Kong, MYao, SYang, Z Li. Growth and metastasis suppression of glioma xenografts expressing exon 4-deletion variant of epidermal growth factor receptor by monoclonal antibody CH12-mediated receptor degradation. FASEB J 2012; 26(1): 73–80 pmid: 21917986
24 RBLuwor, TG Johns, CMurone, HJHuang, WKCavenee, GRitter, LJOld, AW Burgess, AMScott. Monoclonal antibody 806 inhibits the growth of tumor xenografts expressing either the de2-7 or amplified epidermal growth factor receptor (EGFR) but not wild-type EGFR. Cancer Res 2001; 61(14): 5355–5361
pmid: 11454674
25 HJiang, Q Dong, XLuo, BShi, H Wang, HGao, JKong, J Zhang, ZLi. The monoclonal antibody CH12 augments 5-fluorouracil-induced growth suppression of hepatocellular carcinoma xenografts expressing epidermal growth factor receptor variant III. Cancer Lett 2014; 342(1): 113–120 pmid: 24007863
26 MZhou, H Wang, KZhou, XLuo, X Pan, BShi, HJiang, JZhang, KLi, HM Wang, HGao, SLu, M Yao, YMao, HYWang, SYang, J Gu, CLi, ZLi. A novel EGFR isoform confers increased invasiveness to cancer cells. Cancer Res 2013; 73(23): 7056–7067 pmid: 24240702
27 HWang, H Jiang, MZhou, ZXu, S Liu, BShi, XYao, M Yao, JGu, ZLi. Epidermal growth factor receptor vIII enhances tumorigenicity and resistance to 5-fluorouracil in human hepatocellular carcinoma. Cancer Lett 2009; 279(1): 30–38 pmid: 19217205
28 DBalin-Gauthier, JP Delord, PRochaix, VMallard, FThomas, IHennebelle, RBugat, PCanal, CAllal. In vivo and in vitro antitumor activity of oxaliplatin in combination with cetuximab in human colorectal tumor cell lines expressing different level of EGFR. Cancer Chemother Pharmacol 2006; 57(6): 709–718 pmid: 16320055
29 YYang, H Jiang, HGao, JKong, P Zhang, SHu, BShi, P Zhang, MYao, ZLi. The monoclonal antibody CH12 enhances the sorafenib-mediated growth inhibition of hepatocellular carcinoma xenografts expressing epidermal growth factor receptor variant III. Neoplasia 2012; 14(6): 509–518 pmid: 22787432
30 KLiu, H Gao, QWang, LWang, B Zhang, ZHan, XChen, M Han, MGao. Hispidulin suppresses cell growth and metastasis by targeting PIM1 through JAK2/STAT3 signaling in colorectal cancer. Cancer Sci 2018; 109(5): 1369–1381 pmid: 29575334
31 CBaratelli, C Zichi, MDi Maio, MPBrizzi, CSonetto, GVScagliotti, MTampellini. A systematic review of the safety profile of the different combinations of fluoropyrimidines and oxaliplatin in the treatment of colorectal cancer patients. Crit Rev Oncol Hematol 2018; 122: 21–29 pmid: 29458787
32 FMde Man, AKL Goey, RHNvan Schaik, RHJMathijssen, SBins. Individualization of irinotecan treatment: a review of pharmacokinetics, pharmacodynamics, and pharmacogenetics. Clin Pharmacokinet 2018; 57(10):1229–1254
33 KFujita, Y Kubota, HIshida, YSasaki. Irinotecan, a key chemotherapeutic drug for metastatic colorectal cancer. World J Gastroenterol 2015; 21(43): 12234–12248 pmid: 26604633
34 CLTeng, CY Wang, YHChen, CHLin, WL Hwang. Optimal sequence of irinotecan and oxaliplatin-based regimens in metastatic colorectal cancer: a population-based observational study. PLoS One 2015; 10(8): e0135673 pmid: 26273837
35 CMontagut, J Albanell. Mechanisms of acquired resistance to anti-EGF receptor treatment in colorectal cancer. Colorectal Cancer 2012; 1(6): 491–502
36 HKGan, AW Burgess, AHClayton, AMScott. Targeting of a conformationally exposed, tumor-specific epitope of EGFR as a strategy for cancer therapy. Cancer Res 2012; 72(12): 2924–2930 pmid: 22659454
37 JERogers. Patient considerations in metastatic colorectal cancer — role of panitumumab. OncoTargets Ther 2017; 10: 2033–2044 pmid: 28435294
38 MJhawer, S Goel, AJWilson, CMontagna, YHLing, DSByun, SNasser, DArango, JShin, L Klampfer, LHAugenlicht, RPerez-Soler, JMMariadason. PIK3CA mutation/PTEN expression status predicts response of colon cancer cells to the epidermal growth factor receptor inhibitor cetuximab. Cancer Res 2008; 68(6): 1953–1961 pmid: 18339877
39 WZhang, M Gordon, OAPress, KRhodes, DVallböhmer, DYYang, DPark, W Fazzone, ASchultheis, AESherrod, SIqbal, SGroshen, HJLenz. Cyclin D1 and epidermal growth factor polymorphisms associated with survival in patients with advanced colorectal cancer treated with cetuximab. Pharmacogenet Genomics 2006; 16(7): 475–483 pmid: 16788380
40 SKong, CI Amos, RLuthra, PMLynch, BLevin, MLFrazier. Effects of cyclin D1 polymorphism on age of onset of hereditary nonpolyposis colorectal cancer. Cancer Res 2000; 60(2): 249–252
pmid: 10667569
41 BCKoehler, AL Scherr, SLorenz, TUrbanik, NKautz, CElssner, SWelte, JLBermejo, DJäger, HSchulze-Bergkamen. Beyond cell death — antiapoptotic Bcl-2 proteins regulate migration and invasion of colorectal cancer cells in vitro. PLoS One 2013; 8(10): e76446 pmid: 24098503
42 QRQi, ZM Yang. Regulation and function of signal transducer and activator of transcription 3. World J Biol Chem 2014; 5(2): 231–239
pmid: 24921012
[1] Yiming Ma,Ting Xiao,Quan Xu,Xinxin Shao,Hongying Wang. iTRAQ-based quantitative analysis of cancer-derived secretory proteome reveals TPM2 as a potential diagnostic biomarker of colorectal cancer[J]. Front. Med., 2016, 10(3): 278-285.
[2] CAO Jie, LI Wanglin, XIA Jie, TANG Weibiao, WANG Hui, CHEN Xiwen, XIAO Huanqing, LI Yuyuan, CHEN Xiaoping, DU Hong, CHEN Shanming. Absence of FHIT expression is associated with apoptosis inhibition in colorectal cancer[J]. Front. Med., 2007, 1(2): 147-156.
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