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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.
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