Abstract
Anti-EGFR mAb is reported to induce EGFR internalization in colorectal cancer cells. However, the biological relevance of EGFR internalization with anti-EGFR mAb is unknown. Therefore, the relevance of EGFR downregulation with anti-EGFR mAb to antitumor activity in colorectal cancer cells was investigated. Quantification of EGFR on the cell surface before cetuximab treatment was assessed by flow cytometry, and its growth-inhibitory effects were measured by Trypan blue exclusion, in 10 RAS, BRAF wild-type colorectal cancer cell lines, but there was no significant correlation between EGFR number and its growth-inhibitory effect. However, a significant correlation existed between the percentage decrease in the number of EGFRs after cetuximab treatment and its growth-inhibitory effect in those cell lines. Treatment with TGFα, a ligand for EGFR, induced EGFR internalization in colorectal cancer cells, but most EGFRs subsequently recycled to the cell surface, consistent with previous studies. While cetuximab treatment induced EGFR internalization, most receptors subsequently translocated into the late endosome, leading to lysosomal degradation, as revealed by immunoblotting and double immunofluorescence. Cetuximab-sensitive colorectal cancer cells showed greater EGFR internalization, stronger cell growth inhibition, and more augmented apoptotic signals than nonsensitive cells. IHC for EGFR, performed using an EGFR pharmDx Kit (mouse anti-human EGFR mAb clone 2-18C9), in clinical specimens before and after anti-EGFR mAb therapy in 13 colorectal cancer patients showed a significant correlation between the response to anti-EGFR mAb and decreased staining after therapy.Implications: This report clearly demonstrates that anti-EGFR mAb facilitates internalization and subsequent degradation of EGFRs in lysosomes, which is an important determinant of the efficacy of anti-EGFR mAb treatment for colorectal cancer. Mol Cancer Res; 15(10); 1445-54. ©2017 AACR.
Highlights
EGFR represents a unique target in cancer therapy because overexpression of EGFRs has been implicated in the pathogenesis of many malignant tumors, such as head and neck cancer, colorectal cancer, lung cancer, ovarian cancer, cervical cancer, and gastric cancer [1,2,3,4,5,6]
We evaluated the inhibitory effect of cetuximab on cell growth in these 10 RAS, RAF wild-type colorectal cancer cell lines (Fig. 1)
We have shown that the degree of EGFR internalization varies in RAS wild-type colorectal cancer cells and is closely associated with the sensitivity to anti-EGFR antibodies
Summary
EGFR represents a unique target in cancer therapy because overexpression of EGFRs has been implicated in the pathogenesis of many malignant tumors, such as head and neck cancer, colorectal cancer, lung cancer, ovarian cancer, cervical cancer, and gastric cancer [1,2,3,4,5,6]. There are two therapeutic strategies targeting EGFRs: mAbs and tyrosine kinase inhibitors against EGFR. Metastatic colorectal cancer (mCRC), two mAbs targeting EGFR, cetuximab and panitumumab, have been proven to be effective in combination with chemotherapy or as monotherapy [10,11,12,13,14,15]. RAS mutation is the only established biomarker for selection of patients with mCRC. Numerous studies have investigated the association of EGFR molecular events with the response to EGFR mAbs and have demonstrated that the levels of expression or somatic mutations of EGFR did not correlate with clinical responses to cetuximab and panitumumab. The response to EGFR mAbs varies among individuals and cannot be universally expected even in the RAS wild-type mCRC, which presents a significant problem in clinical practice
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