EGFR Downregulation after Anti-EGFR Therapy Predicts the Antitumor Effect in Colorectal Cancer

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 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. 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. .

Colorectal adenocarcinoma‐derived EGFR mutants are oncogenic and sensitive to EGFR‐targeted monoclonal antibodies, cetuximab and panitumumab

“…Cetuximab and panitumumab are FDA approved epidermal growth factor receptor (EGFR)‐targeted monoclonal antibodies that have been widely used in treatment of metastatic colorectal cancer (mCRC) after the failure of first‐line chemotherapy; however, a clinical response can be achieved in only 10~20% of unselected patients . Cetuximab and panitumumab mechanisms of action suggest that they function as antagonists of EGFR ligands through the prevention of ligand‐dependent receptor rearrangement, which is essential for inducing its enzymatic activation, as well as promoting receptor degradation and antibody‐dependent cellular cytotoxicity via the triggering of an immune response . Genomic alterations targeting KRAS , NRAS , BRAF , PIK3C A or ERBB2 , loss of PTEN and amplification of EGFR or ERBB2 are now known to be negative predictors associated with the drug efficacy among CRC patients .…”

“…[1][2][3] Cetuximab and panitumumab mechanisms of action suggest that they function as antagonists of EGFR ligands through the prevention of ligand-dependent receptor rearrangement, which is essential for inducing its enzymatic activation, [4][5][6] as well as promoting receptor degradation and antibody-dependent cellular cytotoxicity via the triggering of an immune response. 7,8 Genomic alterations targeting KRAS, NRAS, BRAF, PIK3CA or ERBB2, loss of PTEN and amplification of EGFR or ERBB2 are now known to be negative predictors associated with the drug efficacy among CRC patients. [9][10][11][12][13][14][15] To date, biomarker discovery has focused on "negative" biomarkers, those which predict likely failure of EGFR antibody treatment, rather than the identification of "positive" markers which identify patients with specific characteristics where EGFR inhibition may be especially likely to succeed.…”

Identification and Characterization of Unique Neutralizing Antibodies to Mouse EGF Receptor

“…5 It has been shown that EGFR is endocytosed upon cetuximab binding and trafficked to late endosomes, leading to lysosomal degradation; in fact, receptor degradation appears to be an important determinant of the antitumor efficacy of cetuximab. 7 We tested whether EGFR undergoes a similar fate after P1X/P2X treatment. Liver organoids from Egfr Em/Em mice were treated with P1X/P2X and then stained with the lysosomal marker LAMP1.…”

Epidermal growth factor receptor‐targeted molecular imaging of colorectal tumors: Detection and treatment evaluation of tumors in animal models

“…Quantitative flow cytometric analysis of EGFR on the cell surface was carried out using mouse antihuman EGFR monoclonal antibody (sc‐120; Santa Cruz Biotechnology, Dallas, TX, USA) and Dako QIFIKIT (Dako, Glostrup, Denmark), as described previously . In brief, cells were incubated with a mouse antihuman EGFR monoclonal antibody.…”

“…Quantitative flow cytometric analysis of EGFR on the cell surface was carried out using mouse antihuman EGFR monoclonal antibody (sc-120; Santa Cruz Biotechnology, Dallas, TX, USA) and Dako QIFIKIT (Dako, Glostrup, Denmark), as described previously. 22 In brief, cells were incubated with a mouse antihuman EGFR monoclonal antibody. After washing the cells with PBS, The cells were observed by confocal laser microscopy (Nikon A1; Nikon, Tokyo, Japan) and bench top fluorescence microscopy (BIOREVO BZ9000; Keyence, Osaka, Japan).…”