The anti–epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab has been approved for the treatment of patients with metastatic colorectal cancer. However, there is currently no reliable marker for response to therapy with the EGFR inhibitors. In this study, we investigated the sensitivity of 10 human colorectal tumor cell lines (DiFi, CCL218, CCL221, CCL225, CCL227, CCL228, CCL231, CCL235, CCL244, and HCT-116) to treatment with our anti-EGFR monoclonal antibody, ICR62, and/or the EGFR tyrosine kinase inhibitor, gefitinib. Of the cells examined, only DiFi contained high levels of constitutively active EGFR and were highly sensitive to treatment with both ICR62 (IC50 = 0.52 nmol/L) and gefitinib (IC50 = 27.5 nmol/L). In contrast, the growth of other tumor cell lines, which contained low levels of the EGFR, HER-2, and pAkt but comparable or even higher basal levels of phosphorylated mitogen-activated protein kinase (pMAPK), were relatively resistant to treatment with both inhibitors. Both ICR62 and gefitinib induced EGFR down-regulation, reduced the basal levels of pEGFR at five known tyrosine residues, pMAPK, and pAkt, and increased the sub-G1 population in DiFi cells. However, treatment with a combination of ICR62 and gefitinib neither sensitized colorectal tumor cells that were insensitive to treatment with the single agent nor enhanced the growth-inhibitory effect of the single agent in DiFi cells. These results indicate that basal levels of pMAPK and pAkt are not good indicators of response to the EGFR inhibitors in colorectal cancer cells and dual targeting of the EGFR by a combination of ICR62 and gefitinib is not superior to treatment with a single agent. (Cancer Res 2006; 66(15): 7708-15)

Colorectal cancer is one of the major types of cancer worldwide, in terms of both morbidity and mortality. Despite incremental improvements in adjuvant therapy over the past decade, the outcomes of treatment for locally advanced and metastatic colorectal cancer remains disappointing with 5-year survival rates of <10% in patients with metastatic disease (1).

Since the early 1980s, aberrant expression of the epidermal growth factor receptor (EGFR) has been reported in a wide range of human epithelial malignancies, including colorectal cancer, and in some studies, EGFR expression was shown to be associated with a poor prognosis and resistance to conventional therapies (2, 3). These discoveries have lead to the strategic development of novel agents targeting the EGFR for the treatment of human malignancies (4). Of these, the two most clinically advanced strategies are the monoclonal antibodies (mAb), which block the extracellular ligand-binding domain, and small molecule tyrosine kinase inhibitors (TKI), which target ATP-binding sites located in the intracellular tyrosine kinase domain of the EGFR (3, 5, 6). Three such agents, i.e., the anti-EGFR mAb cetuximab (Erbitux), and the TKIs, gefitinib and erlotinib, have now been approved for the treatment of patients with metastatic colorectal cancer and non–small cell lung cancer, respectively (5, 6). Despite objective responses to the EGFR inhibitors in a small fraction of patients, there has been no clear association between the levels of tumor EGFR expression and response to treatment with the EGFR inhibitors (713). Several studies have suggested that the expression of other growth factor receptors (e.g., HER-2, HER-3, and IGF-IR), the presence of somatic mutation in exons 18 to 21 of the EGFR gene, or increased numbers of the EGFR gene might be associated with responses to treatment with the EGFR inhibitors (3, 1422).

Because anti-EGFR mAbs and the EGFR TKIs target two distinct epitopes on the EGFR, the potential therapeutic advantage of using a combination of the two approaches for cancer therapy has been highlighted in several studies (23, 24). Indeed, in two recent studies, cotargeting of the EGFR with a combination of cetuximab and gefitinib or erlotinib was shown to be superior over the targeting of tumor cells with the single agent (25, 26). More recently, the combination of cetuximab with gefitinib was reported to induce an antagonistic effect in EGFR-positive tumor cells (27).

We have previously reported the development of a panel of mAbs against the extracellular domain of the EGFR (28). Of these, mAb ICR62 blocks the binding of ligands to the EGFR and subsequent phosphorylation of the EGFR, and inhibits the growth of several EGFR-overexpressing (e.g., head and neck, breast, and vulva) cell lines both in vitro and in vivo (29). In addition, mAb ICR62 has been shown to inhibit the growth in vivo of EGFRvIII-expressing tumors by mediating antibody-dependent cellular cytotoxicity and localization to metastatic lesions in cancer patients 24 hours after administration (30, 31). The aim of this study was to evaluate the sensitivity of a panel of human colorectal tumor cell lines to treatment with anti-EGFR mAb ICR62 used alone and in combination with gefitinib. We also examined the effects of mAb ICR62 and gefitinib on the cellular location of the EGFR, cell cycle distribution, levels of EGFR phosphorylation at five known tyrosine residues (1173, 1148, 1086, 1068, and 845), and downstream effectors such as ERK1/2 and Akt in the colorectal tumor cells. The relationship between growth factor receptor expression (EGFR and HER-2) in colorectal tumor cells and growth inhibition by these EGFR inhibitors was also investigated.

Tumor cell lines. The human colorectal cancer cell lines CCL218/HT-29, CCL221/DLD-1, CCL225/HCT-15, CCL227/SW620, CCL228/SW480, CCL231/SW48, CCL235/SW837, and CCL244/HCT-8/HRT-18 were purchased from the American Type Culture Collection (Manassas, VA) and CCL247/HCT-116 from European Collection of Cell Culture (Porton Down, United Kingdom). Other human tumor cell lines used in this study included DiFi (colon) cells, which express 4.8 × 106 EGFR/cell (32), SKBR3 (breast), MCF-7 (breast), and HN5 (head and neck) cells, which overexpress HER-2, IGF-IR, and EGFR, respectively (3335). All cell lines were cultured in DMEM (Sigma-Aldrich Company, Ltd., Dorset, United Kingdom), supplemented with 10% FCS (GIBCO Cell Culture Systems, Invitrogen Ltd., Paisley, United Kingdom), penicillin (50 μg/mL), streptomycin (50 μg/mL), and neomycin (100 μg/mL; Life Technologies, United Kingdom), and were maintained at 37°C in a humidified atmosphere with 5% CO2.

Antibodies and EGFR inhibitors. The rat mAbs, ICR62 (IgG2b) and ICR16 (IgG2a), were raised against the external domain of the EGFR on the breast (MDA-MB468) and head and neck (HN5) carcinoma cell lines, respectively (28, 29). The mouse mAbs, HM50.67A and HM43.16B, were raised against the external domain of the HER-2 and EGFR, respectively.3

3

H. Modjtahedi, S. Eccles, D.K. Moscatello, and A.J. Wong, unpublished data.

The mouse anti-EGFR mAb clone F4 and rabbit anti-β-actin polyclonal antibody were purchased from Sigma-Aldrich. Antibodies to phosphotyrosine (P-Tyr-100), pEGFR (Tyr1068), and phosphorylated mitogen-activated protein kinase (pMAPK; Thr202/Tyr204) were purchased from New England Biolabs UK, Ltd. (Hitchin, United Kingdom). Antibodies to tyrosine phosphorylation–specific EGFR (Tyr1173, Tyr1148, Tyr1086, and Tyr845), and Ser473 phosphorylation–specific Akt were purchased from Biosource Europe S.A. (Belgium). Commercial secondary antibodies used in this study included FITC-conjugated goat anti-mouse IgG (Southern Biotechnology Associates Inc., Birmingham, AL), and FITC-conjugated rabbit anti-rat IgG (Serotec Ltd., Oxford, United Kingdom). The EGFR TKI gefitinib (Iressa/ZD1839) was kindly provided by AstraZeneca (Macclesfield, United Kingdom).

Flow cytometry. The cell surface expression of growth factor receptors was determined using fluorescence-activated cell sorting (FACS) analysis. Approximately 1 × 106 tumor cells in 1 mL of DMEM/2% FCS were incubated with control medium (i.e., no treatment) or 10 μg of the anti-EGFR mAb HM43.16B or the anti-HER-2 mAb HM50.67A for 1 hour by rotation at 4°C. Tumor cells were washed thrice by centrifugation for 5 minutes at 1,200 rpm (264 × g) and resuspension in DMEM/2% FCS, prior to incubation with FITC-conjugated goat anti-mouse IgG secondary antibody. Following rotation for 1 hour at 4°C, tumor cells were washed three more times and the final cell pellet was resuspended in 0.5 mL of FACS Flow buffer (Becton Dickinson UK, Ltd., Oxford, United Kingdom). A minimum of 10,000 events were recorded by excitation with an argon laser at 488 nm, and analyzed using the FL-1 detector (FITC detector; 525 nm) of a Beckman Coulter Epics XL flow cytometer (Becton Dickinson) and CellQuest software.

Cell cycle distribution analysis. Approximately 1 × 106 tumor cells were seeded into 25 cm2 culture flasks containing 15 mL of DMEM/2% FCS plus mAb ICR62 (156 nmol/L), gefitinib (300 nmol/L), or control medium. Following incubation for 5 days at 37°C, the supernatants were collected and the adherent cells were trypsinized and pooled together with the cell supernatant. Cells were then washed thrice in cold PBS and the final cell pellet was resuspended in 200 μL of cold PBS and fixed in 1 mL of cold 70% ethanol (in PBS). Following incubation overnight at 4°C, tumor cells were washed once in PBS and incubated with 500 μL of PBS containing 1 mg/mL RNase and 10 μg/mL propidium iodide for 30 minutes at 37°C in the dark. Propidium iodide–stained tumor cells were excited at 488 nm and analyzed using the FL-3 detector (620 nm) of a Beckman Coulter Epics XL flow cytometer (Becton Dickinson).

Growth inhibition studies. The effect of EGFR inhibitors on the growth of human tumor cell lines was investigated using a colorimetric assay, as described previously (28). Briefly, tumor cells were seeded at a density of 5 × 103/well in 100 μL of DMEM containing 2% to 10% FCS in a 96-well plate. Following 3-hours of incubation at 37°C, 100 μL aliquots of ICR62 and/or gefitinib were added to triplicate wells and the cells were incubated for 6 to 12 days, depending on the cell line, at 37°C until the cells in the wells containing control medium were confluent. Tumor cells were then fixed with glutaraldehyde, washed in distilled water, air-dried, and stained with 0.05% methylene blue. The absorbance of each well was measured at 620 nm using a Labsystems MultiSkan RC plate reader (Thermo Electron Corporation, Basingstoke, United Kingdom). In order to establish the initial number of cells plated, an extra plate of cells was set up and similarly processed after 3 hours of incubation at 37°C without the inhibitors.

Immunofluorescence staining of tumor cells. The location of the EGFR following treatment with the EGFR inhibitors was investigated using immunofluorescence staining. For this purpose, tumor cells in DMEM/10% FCS were grown to near-confluence in 16-well Lab-Tek Chamber Slides (Nalge, Hereford, United Kingdom) coated with fibronectin (1/40 in PBS; Sigma-Aldrich). Following washes in DMEM/0.1% FCS, cells were incubated with ICR62 (100 nmol/L), gefitinib (200 nmol/L), or control medium for 1 or 24 hours at 37°C. Tumor cells were washed thrice in PBS, fixed for 15 minutes at room temperature in 4% paraformaldehyde, washed an additional three times in PBS, and permeabilized for 15 minutes at room temperature in 0.5% Triton X-100 (Sigma-Aldrich). Following further washes in PBS, tumor cells were incubated in the presence of mAb ICR62 (20 μg/mL) or control medium for 1 hour at 4°C. The bound antibody was detected following incubation with rabbit anti-rat FITC-conjugated secondary antibody (1/400; Serotec) for 30 minutes at 4°C. Following three more washes, cells were incubated with 2 μg/mL of Hoechst 33258 nuclear stain (Sigma-Aldrich) for 5 minutes at room temperature, mounted with H-1000 mounting medium (Vector Laboratories, Ltd., Peterborough, United Kingdom), and examined for fluorescence under a Zeiss Axiovert 100 microscope (Carl Zeiss, Ltd., Welwyn Garden City, United Kingdom) using the Leica Qwin software.

Western blot analysis. The effect of anti-EGFR mAb ICR62 and/or gefitinib on the downstream signaling of human tumor cells was investigated by Western blotting analysis. Tumor cells were grown to near-confluence in six-well tissue culture plates (Greiner Bio-One, Gloucestershire, United Kingdom) containing 5 mL of growth medium. The cell monolayers were washed once and then incubated in 5 mL of DMEM/0.1% FCS containing control medium, ICR62 (400 nmol/L) and/or gefitinib (400 nmol/L) for 24 hours at 37°C, prior to stimulation with 10 nmol/L of EGF (R&D Systems, Oxon, United Kingdom) for 15 minutes at 37°C. Tumor cells were washed with PBS and then solubilized with 400 μL of LDS sample buffer (Invitrogen) supplemented with 4 μL of protein inhibitor cocktail containing 104 mmol/L of AEBSF, 80 μmol/L of aprotinin, 2 mmol/L of leupeptin, 4 mmol/L of bestatin, 1.5 mmol/L of pepstatin A, and 1.4 mmol/L of E-64 (Sigma-Aldrich). The cell lysates were heated at 72°C for 10 minutes and the viscosity was reduced by several passages through a 25 × 5/8-gauge needle. Equal amounts of cell lysate were separated on 4% to 12% Bis-Tris gels (Invitrogen) using the XCell II Surelock Mini-Cell system (Invitrogen) and transferred to polyvinylidene difluoride membranes using the XCell II Mini-Cell Blot Module kit (Invitrogen). The polyvinylidene difluoride membranes were incubated with primary antibody for 1 hour at room temperature, and the specific signals were detected using the WesternBreeze chemiluminescence kit (Invitrogen).

Expression of cell surface EGFR family members (EGFR and HER-2) and basal phosphorylation levels of EGFR, MAPK, and Akt in a panel of human colorectal tumor cell lines. The cell surface expression of the EGFR and HER-2 detected by FACS analysis in the panel of human colorectal cell lines in reference to control cell lines is summarized in Table 1. Similar to HN5 head and neck cancer cells, the DiFi cell line was found to express high levels of the EGFR, with a mean fluorescence intensity (MFI) of 169.6, whereas the levels of EGFR were much lower in the remaining nine colorectal cancer cell lines (MFI range, 9.0-21.1). Compared with the level of HER-2 overexpression in SKBR3 breast cancer cells (MFI, 206.1; Table 1), the expression of HER-2 was found to be much lower in this panel of human colorectal cancer cell lines (MFI range, 9.0-40.1).

Table 1.

The cell surface expression of EGFR and HER2 measured by FACS analysis in colorectal tumor cell lines and respective positive control cell lines

Cell lineMFI ± SD
ControlEGFRHER-2
DiFi 3.6 ± 0.34 169.6 ± 0.34 19.6 ± 0.96 
HCT116 2.3 ± 0.19 9.0 ± 0.2 9.0 ± 0.5 
CCL218 2.1 ± 0.21 8.4 ± 0.1 14.7 ± 0.99 
CCL221 3.7 ± 0.36 21.1 ± 0.17 32.6 ± 1.3 
CCL225 4.0 ± 0.33 19.8 ± 0.1 40.1 ± 1.52 
CCL227 2.9 ± 0.31 19.2 ± 0.08 15.5 ± 0.84 
CCL228 3.4 ± 0.24 12.2 ± 0.13 11.8 ± 0.64 
CCL231 3.5 ± 0.36 20.4 ± 0.63 21.8 ± 0.09 
CCL235 3.3 ± 0.22 17.2 ± 0.09 17.2 ± 0.55 
CCL244 4.0 ± 0.28 13.2 ± 0.01 20.1 ± 0.12 
HN5 4.0 ± 0.28 186.1 ± 11.4 12.3 ± 0.12 
SKBR3 2.5 ± 0.09 11.2 ± 0.17 206.1 ± 5.6 
MCF7 4.1 ± 0.13 20.9 ± 0.04 17.8 ± 0.34 
Cell lineMFI ± SD
ControlEGFRHER-2
DiFi 3.6 ± 0.34 169.6 ± 0.34 19.6 ± 0.96 
HCT116 2.3 ± 0.19 9.0 ± 0.2 9.0 ± 0.5 
CCL218 2.1 ± 0.21 8.4 ± 0.1 14.7 ± 0.99 
CCL221 3.7 ± 0.36 21.1 ± 0.17 32.6 ± 1.3 
CCL225 4.0 ± 0.33 19.8 ± 0.1 40.1 ± 1.52 
CCL227 2.9 ± 0.31 19.2 ± 0.08 15.5 ± 0.84 
CCL228 3.4 ± 0.24 12.2 ± 0.13 11.8 ± 0.64 
CCL231 3.5 ± 0.36 20.4 ± 0.63 21.8 ± 0.09 
CCL235 3.3 ± 0.22 17.2 ± 0.09 17.2 ± 0.55 
CCL244 4.0 ± 0.28 13.2 ± 0.01 20.1 ± 0.12 
HN5 4.0 ± 0.28 186.1 ± 11.4 12.3 ± 0.12 
SKBR3 2.5 ± 0.09 11.2 ± 0.17 206.1 ± 5.6 
MCF7 4.1 ± 0.13 20.9 ± 0.04 17.8 ± 0.34 

The basal level of phosphorylated EGFR, pMAPK, and phosphorylated Akt were determined in the panel of colorectal tumor cell lines using Western blot analysis (Fig. 1A). DiFi cells were found to contain a very high basal level of tyrosine phosphorylated EGFR, which is indicative of high EGFR tyrosine kinase activity. In contrast, the other nine colorectal tumor cell lines were found to have nearly undetectable levels of tyrosine-phosphorylated EGFR and Ser473-phosphorylated Akt but show comparable or even higher levels of pMAPK, particularly in CCL218, CCL221, CCL227, CCL228, and CCL231 (Fig. 1A).

Figure 1.

The basal levels of pEGFR, pMAPK, and pAkt in human colorectal tumor cell lines (A). Tumor cells growing under normal conditions (10% FCS) were lysed, and 50 μg of each protein lysate was analyzed by Western blot analysis for the levels of tyrosine-phosphorylated EGFR (170 kDa), pMAPK, pAkt, and β-actin. The effect doubling dilutions of mAb ICR62 or gefitinib on the growth of EGFR-overexpressing DiFi cells (B). Tumor cells were grown in DMEM/2% FCS containing mAb ICR62, gefitinib, or control medium, until cells in wells containing control medium were confluent. Tumor cell proliferation was calculated as a percentage of control cell growth, as described in “Materials and Methods.” Immunofluorescence staining of the EGFR in DiFi cells growth-inhibited by ICR62 (100 nmol/L), gefitinib (200 nmol/L), or following treatment with control medium (C). The effect of the highest concentrations of mAb ICR62 or gefitinib on the growth of human colorectal tumor and positive control cells (D). Columns, mean of triplicate values; bars, ±SD.

Figure 1.

The basal levels of pEGFR, pMAPK, and pAkt in human colorectal tumor cell lines (A). Tumor cells growing under normal conditions (10% FCS) were lysed, and 50 μg of each protein lysate was analyzed by Western blot analysis for the levels of tyrosine-phosphorylated EGFR (170 kDa), pMAPK, pAkt, and β-actin. The effect doubling dilutions of mAb ICR62 or gefitinib on the growth of EGFR-overexpressing DiFi cells (B). Tumor cells were grown in DMEM/2% FCS containing mAb ICR62, gefitinib, or control medium, until cells in wells containing control medium were confluent. Tumor cell proliferation was calculated as a percentage of control cell growth, as described in “Materials and Methods.” Immunofluorescence staining of the EGFR in DiFi cells growth-inhibited by ICR62 (100 nmol/L), gefitinib (200 nmol/L), or following treatment with control medium (C). The effect of the highest concentrations of mAb ICR62 or gefitinib on the growth of human colorectal tumor and positive control cells (D). Columns, mean of triplicate values; bars, ±SD.

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Growth response of human colorectal tumor cells to treatment with anti-EGFR mAb ICR62 and/or gefitinib. The effect of the EGFR inhibitors on the proliferation of the human colorectal tumor cells was examined by a colorimetric assay. Of the 10 colorectal tumor cell lines, only DiFi cells were highly sensitive to the EGFR inhibitors (Fig. 1B). As the results in Fig. 1B show, complete growth inhibition of DiFi cells by mAb ICR62 was achieved at a concentration of 3.2 nmol/L (IC50 = 0.52 nmol/L). Gefitinib also inhibited the growth of DiFi cells, with complete growth inhibition at 87.4 nmol/L (IC50 = 27.5 nmol/L), and DiFi cells growth-inhibited by both EGFR inhibitors were still EGFR-positive (Fig. 1C). In contrast to DiFi cells and control EGFR-overexpressing HN5 cells, the remaining colorectal tumor cell lines were not sensitive to treatment with either of the EGFR inhibitors (Fig. 1D). Unlike the EGFR-overexpressing cells which were highly sensitive to treatment with both EGFR inhibitors, the growth of HER-2-overexpressing SKBR3 cells was only inhibited by gefitinib. At 500 nmol/L, gefitinib inhibited the growth of SKBR3 cells by 82% (Fig. 1D).

A recent study has reported that the combination of anti-EGFR mAb cetuximab and gefitinib produced synergistic growth inhibition of DiFi cells (26). Therefore, we investigated whether a combination of ICR62 and gefitinib would achieve better growth-inhibitory responses in the panel of human colorectal tumor cell lines. However, using similar exposure times for treatments and similar concentrations of the EGFR inhibitors described in that study (26), we found no substantial increase in growth inhibition of DiFi cells following the combined treatment with mAb ICR62 and gefitinib. Because DiFi cells are highly sensitive to treatment with mAb ICR62, a caveat is that a potential synergism might be observed if lower concentrations of mAb ICR62 were used. Our primary interest was to determine whether a combination of mAb ICR62 and gefitinib would sensitize the panel of colorectal tumor cell lines that are not sensitive to the either agent when used alone. Disappointingly, we found no enhancement of growth inhibition in this panel of cell lines following treatment with a combination of mAb ICR62 and gefitinib (Fig. 2B and C).

Figure 2.

The effect of doubling dilutions of mAb ICR62 in combination with gefitinib on the growth of EGFR-overexpressing (MFI = 169) DiFi cells (A) or low EGFR–expressing (MFI = 13) CCL244 cells (B). The effect of mAb ICR62 (200 nmol/L) in combination with gefitinib (200 nmol/L) on the growth of human colorectal tumor and positive control EGFR-overexpressing (HN5), HER-2 overexpressing (SKBR3) cells, or MCF-7 cells expressing low levels of EGFR and HER-2 (C), as described in “Materials and Methods.” In addition, DiFi cells were incubated in DMEM/10% FCS for 24 hours at 37°C prior to treatment with mAb ICR62 (0.5, 5, and 50 nmol/L) and/or gefitinib (0.1, 1, and 10 μmol/L) for 72 hours at 37°C (D) according to the method described in ref. (26). Points, mean of triplicate values; bars, ±SD.

Figure 2.

The effect of doubling dilutions of mAb ICR62 in combination with gefitinib on the growth of EGFR-overexpressing (MFI = 169) DiFi cells (A) or low EGFR–expressing (MFI = 13) CCL244 cells (B). The effect of mAb ICR62 (200 nmol/L) in combination with gefitinib (200 nmol/L) on the growth of human colorectal tumor and positive control EGFR-overexpressing (HN5), HER-2 overexpressing (SKBR3) cells, or MCF-7 cells expressing low levels of EGFR and HER-2 (C), as described in “Materials and Methods.” In addition, DiFi cells were incubated in DMEM/10% FCS for 24 hours at 37°C prior to treatment with mAb ICR62 (0.5, 5, and 50 nmol/L) and/or gefitinib (0.1, 1, and 10 μmol/L) for 72 hours at 37°C (D) according to the method described in ref. (26). Points, mean of triplicate values; bars, ±SD.

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Mechanistic insights of treatment of DiFi cells with mAb ICR62 or gefitinib. The changes in the cell surface level of EGFR in DiFi cells following treatment with ICR62 or gefitinib were detected using immunofluorescent staining and the results are presented in Fig. 3A. Following 1 hour of treatment of DiFi cells with the EGFR inhibitors, there were no major changes in the level of cell surface EGFR (data not shown). A substantial loss of membranous EGFR was observed 24 hours after treatment with anti-EGFR mAb ICR62, and also with ICR16, another anti-EGFR mAb (Fig. 3B and C). Interestingly, there was also a noticeable reduction in the level of cell surface EGFR in DiFi cells following treatment with gefitinib (Fig. 3D), which targets the intracellular domain of the EGFR. However, the extent of reduction of cell surface EGFR by gefitinib in DiFi cells was not as great as that achieved with the anti-EGFR mAbs, ICR62 and ICR16.

Figure 3.

Cellular location of EGFR in DiFi cells following treatment with anti-EGFR mAb or EGFR TKI. Near-confluent DiFi cells were cultured for 24 hours in DMEM/0.1% FCS containing control medium (A), 100 nmol/L of mAbs ICR16 (B), ICR62 (C), or 200 nmol/L of gefitinib (D). DiFi cells were permeabilized with 0.5% Triton X-100, followed by sequential incubation with rat mAb ICR62 and FITC-conjugated anti-rat secondary antibody as described in “Materials and Methods.” Left, FITC staining; right, Hoechst 33258 nuclear staining.

Figure 3.

Cellular location of EGFR in DiFi cells following treatment with anti-EGFR mAb or EGFR TKI. Near-confluent DiFi cells were cultured for 24 hours in DMEM/0.1% FCS containing control medium (A), 100 nmol/L of mAbs ICR16 (B), ICR62 (C), or 200 nmol/L of gefitinib (D). DiFi cells were permeabilized with 0.5% Triton X-100, followed by sequential incubation with rat mAb ICR62 and FITC-conjugated anti-rat secondary antibody as described in “Materials and Methods.” Left, FITC staining; right, Hoechst 33258 nuclear staining.

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We next examined the changes in cell cycle distribution of DiFi cells after treatment with anti-EGFR mAb ICR62 and gefitinib using FACS analysis. As expected, treatment of DiFi cells with mAb ICR62 or gefitinib increased the proportion of DiFi cells in the sub-G1 phase of the cell cycle from 2% to 41% and 57%, respectively (Fig. 4A and B). This was accompanied with a reduction in the proportion of cells in the G1, S, and G2-M phases of the cell cycle. For example, treatment of DiFi cells with mAb ICR62 and gefitinib for 5 days decreased the proportion of cells in G1 from 60%, to 45%, and 30%, respectively (Fig. 4). However, the growth-inhibitory effect of both mAb ICR62 and gefitinib on DiFi cells was reversible (Fig. 4C).

Figure 4.

The effect of EGFR inhibitors on the cell cycle distribution of EGFR-overexpressing DiFi cells (A and B). Tumor cells were incubated for 5 days in DMEM/2% FCS containing mAb ICR62 (156 nmol/L), gefitinib (300 nmol/L), or control medium. After the treatment, the cells were harvested, and analyzed for DNA content using FACS analysis, as described in “Materials and Methods.” The DNA histograms for DiFi cells treated with control medium, ICR62, or gefitinib (A), and the percentage ±SD of treated DiFi cells in the sub-G0/G1, G1, S, and G2-M phases of the cell cycle (B). The reversibility of growth arrest by the EGFR inhibitors (C). DiFi cells were cultured in DMEM/2% FCS containing mAb ICR62 (200 nmol/L) or gefitinib (400 nmol/L) continuously for 5 days or 10 days, or for 5 days, followed by another 5 days in inhibitor-free medium. Columns, mean of triplicate values; bars, ±SD.

Figure 4.

The effect of EGFR inhibitors on the cell cycle distribution of EGFR-overexpressing DiFi cells (A and B). Tumor cells were incubated for 5 days in DMEM/2% FCS containing mAb ICR62 (156 nmol/L), gefitinib (300 nmol/L), or control medium. After the treatment, the cells were harvested, and analyzed for DNA content using FACS analysis, as described in “Materials and Methods.” The DNA histograms for DiFi cells treated with control medium, ICR62, or gefitinib (A), and the percentage ±SD of treated DiFi cells in the sub-G0/G1, G1, S, and G2-M phases of the cell cycle (B). The reversibility of growth arrest by the EGFR inhibitors (C). DiFi cells were cultured in DMEM/2% FCS containing mAb ICR62 (200 nmol/L) or gefitinib (400 nmol/L) continuously for 5 days or 10 days, or for 5 days, followed by another 5 days in inhibitor-free medium. Columns, mean of triplicate values; bars, ±SD.

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Finally, we examined the effects of mAb ICR62 and/or gefitinib on basal and EGF-stimulated levels of phosphorylated EGFR, as well as pMAPK and Akt in DiFi cells. Five selected tyrosine sites of the EGFR (1173, 1148, 1086, 1068, and 845) showed high levels of basal phosphorylation. The levels of total EGFR protein and phosphorylation of the five EGFR tyrosine residues were dramatically decreased after 24-hour treatment of DiFi cells with ICR62 and gefitinib. However, at 400 nmol/L, mAb ICR62 was not as effective as gefitinib at reducing total EGFR proteins and the basal level of EGFR tyrosine-phosphorylation (Fig. 5). Treatment with EGF increased the levels of tyrosine phosphorylation of EGFR at each of the five sites and this was also inhibited more effectively by gefitinib than by mAb ICR62 (Fig. 5).

Figure 5.

The phosphorylation status of EGFR, MAPK, and Akt in DiFi cells treated with anti-EGFR mAb and/or EGFR TKI. DiFi cells were cultured to near-confluence in DMEM containing 10% FCS, then treated in DMEM/0.1% FCS containing mAb ICR62 (400 nmol/L), and/or gefitinib (400 nmol/L) for 24 hours at 37°C. Cells were then incubated without growth factor or in 10 nmol/L of EGF for 15 minutes at 37°C. The treated cells were lysed and equal amounts of cell lysate were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies specific for the molecule of interest. The results are representative of at least two independent experiments.

Figure 5.

The phosphorylation status of EGFR, MAPK, and Akt in DiFi cells treated with anti-EGFR mAb and/or EGFR TKI. DiFi cells were cultured to near-confluence in DMEM containing 10% FCS, then treated in DMEM/0.1% FCS containing mAb ICR62 (400 nmol/L), and/or gefitinib (400 nmol/L) for 24 hours at 37°C. Cells were then incubated without growth factor or in 10 nmol/L of EGF for 15 minutes at 37°C. The treated cells were lysed and equal amounts of cell lysate were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies specific for the molecule of interest. The results are representative of at least two independent experiments.

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Figure 5 shows that both MAPK and Akt were phosphorylated in DiFi cells upon EGF stimulation and the levels of pMAPK and Akt were reduced following treatment with ICR62 and gefitinib (Fig. 5). Interestingly, whereas gefitinib reduced EGF-induced Akt phosphorylation in DiFi cells, there seems to be an increase in Akt phosphorylation by mAb ICR62 in the EGF-stimulated cells (Fig. 5). Although, mAb ICR62 and gefitinib as single agents did not prevent EGF-induced phosphorylation of MAPK, a complete reduction of EGF-induced phosphorylation of MAPK was achieved using a combination of mAb ICR62 and gefitinib (Fig. 5).

The aberrant expression of the EGFR in human malignancies and its association with poor prognosis in some patients has resulted in the strategic development of novel agents for targeting the EGFR in human malignancies. Of the EGFR inhibitors, the U.S. Food and Drug Administration has approved the EGFR TKIs gefitinib and erlotinib for the treatment of patients with NSCLC, and the anti-EGFR mAb cetuximab has gained approval for the treatment of patients with colorectal cancer, and more recently (March 1, 2006), head and neck cancer (3, 4, 36). However, a major challenge is the lack of reliable predictive factors for response to therapy with such inhibitors (3, 6, 7). In some patients, in particular, those with NSCLC, the presence of activating mutations in exons 18 to 21 of the EGFR tyrosine kinase domain have been associated with improved response to therapy with the EGFR TKIs (17, 18, 37, 38). However, no association has been reported between the presence of activating EGFR mutations and response to anti-EGFR mAb cetuximab in NSCLC cell lines (38), and the presence of activating EGFR mutation in exons 18 to 21 was found to be rare in human colorectal tumor cell lines and human colorectal tumor specimens (20, 21, 3941). There is, therefore, a need for further study on the underlying mechanism of sensitivity or resistance of human colorectal tumor cells to treatment with anti-EGFR antibodies or the EGFR TKIs and the therapeutic advantages of the EGFR inhibitors when used in combination and/or with other types of therapeutic agents (3, 6).

In this study, we examined the sensitivity of a panel of human colorectal tumor cell lines to treatment with anti-EGFR mAb ICR62 and/or gefitinib and the association between the expression of EGFR and HER-2, and response to the EGFR inhibitors. Of the 10 human colorectal tumor cell lines examined in this study, only DiFi cells overexpressed the EGFR, contained high levels of constitutively active EGFR, and were highly sensitive to growth inhibition by both ICR62 and gefitinib (Figs. 1 and 2; Table 1). Similar to our results, some of the low EGFR–expressing cells used in this study were also found to be relatively resistant to treatment with cetuximab or gefitinib (21, 26, 39, 4244). The high basal level of pMAPK in the human colorectal tumor cells with low levels of EGFR (Fig. 1A; Table 1), suggests that receptors other than the EGFR or HER-2 may be responsible for activation of the MAPK in these cells. This may explain why such tumor cells were insensitive to treatment with the EGFR inhibitors. In addition, the great majority of human tumor cells lines with EGFR overexpression (e.g., DiFi, HN5, A431, HSC-1, and HSC-2) have been shown to be highly sensitive to growth inhibition by anti-EGFR mAbs or the EGFR TKIs (Figs. 1 and 2; Table 1; refs. 26, 34, 45). Taken together, these results suggest that overexpression of the EGFR is a major indictor of response (i.e., growth inhibition) to the EGFR inhibitors. The lack of correlation between EGFR expression and response to the EGFR inhibitors reported in some clinical studies, such as the studies reporting response to cetuximab in patients with EGFR-negative colorectal tumors (10, 46) may, therefore, lie in the technical difficulties associated with accurate measurement of EGFR expression by immunohistochemistry (47, 48).

Gefitinib has previously been shown to inhibit the growth of the HER-2-overexpressing SKBR3 cells in culture (9, 15). In this study, unlike ICR62 (Fig. 1D), gefitinib inhibited the growth of SKBR3 cells, indicating that the mechanisms of action of ICR62 and gefitinib are not identical. In addition, because anti-EGFR mAbs and the EGFR TKI target different domains of the EGFR, we next examined the effect of cotargeting the EGFR with mAb ICR62 and gefitinib in colorectal tumor cells, in particular, those which were not sensitive to treatment with either single agent alone. As shown in Fig. 2A-C, combined treatment with ICR62 and gefitinib was not superior to treatment with the single agent at inhibiting the growth of human colorectal tumor cell lines in vitro. This was seen in cells with both high and low levels of EGFR expression (Fig. 2A-C) and is in contrast with the results of two recent studies (25, 26). In one study, the combination of cetuximab (up to 50 nmol/L) and gefitinib (up to 10 μmol/L) synergistically inhibited the growth of cells with moderate to high EGFR expression (e.g., A431, DiFi, and DU-145), but not cells with low or undetectable levels of EGFR expression (BT-474, SKBR3, and MDA-MB-435S; ref. 26). Interestingly, even using the same concentrations of the EGFR inhibitors and incubation times as those used by Matar and colleagues (26), the combination of mAb ICR62 (and also anti-EGFR mAb ICR16; data not shown) with gefitinib was not found to be synergistic in DiFi cells (Fig. 2D). One possible interpretation is that the synergistic growth-inhibition of DiFi and other tumor cells shown by others (25, 26) is unique to the anti-EGFR mAb cetuximab when used in combination with gefitinib. A recent crystallographic study revealed that cetuximab interacts exclusively with domain III of the extracellular domain of EGFR, partially occluding the ligand-binding region on this domain and sterically preventing the receptor from adopting the extended conformation required for dimerization (49), whereas the epitopes that ICR62 binds remain to de determined. It is therefore possible that the epitopic binding difference between cetuximab and ICR62 may be attributed to the different results. However, in a more recent study, the combination of gefitinib and cetuximab was found to be antagonistic in CAL33 (head and neck) and CAL39 (vulvar) cells, and this was accompanied by an increase in cellular expression of the EGFR (27). These conflicting results raise cautions and demands for further investigation of potential therapeutic benefit by dual targeting of the EGFR with anti-EGFR mAbs and the EGFR TKIs. In addition, because anti-EGFR mAbs such as cetuximab or ICR62 could inhibit the growth of EGFR-expressing tumor cells via immunologic mechanisms, such as antibody-dependent cellular cytotoxicity (26, 31), the results of ongoing clinical trials should unravel whether such combinations are antagonistic or associated with more side effects in patients with cancer (50).

In this study, both ICR62 and gefitinib reversibly inhibited the growth of EGFR-overexpressing DiFi cells in culture and this was accompanied by the down-regulation of membranous EGFR (Figs. 1, and 3-5). Like mAb ICR62 in this study, cetuximab was found to be more effective than gefitinib at inducing down-regulation of the EGFR in tumor cells (27). In addition to down-regulation of the EGFR, both ICR62 and gefitinib reduced the basal levels of EGFR phosphorylation at five known tyrosine residues, as well as pMAPK and pAkt in DiFi cells (Fig. 5), suggesting that a reduction in the basal levels of pEGFR, pMAPK, and pAkt in the EGFR-overexpressing tumor cells is an indicator of response (i.e., growth inhibition) to both inhibitors. However, there were high basal levels of pMAPK in colorectal tumor cells which expressed low levels of EGFR and HER-2 and such cells were resistant to the EGFR inhibitors (Figs. 1 and 2). Therefore, these results suggest that receptors other than EGFR and HER-2 may be responsible for the activation of MAPK in such tumor cells and that the measurement of basal pMAPK alone is not a good indicator of response to treatment with the EGFR inhibitors when used alone or in combination.

In summary, in this study we have shown that with the exception of EGFR overexpression in DiFi cells, which were established from a patient with FAP, overexpression of the EGFR or HER-2 is not common in human colorectal tumor cells (Table 1). Only DiFi cells which overexpressed the EGFR and contained a constitutively active EGFR were highly sensitive to treatment with either ICR62 or gefitinib. Treatment with a combination of ICR62 and gefitinib did not enhance the growth-inhibitory effect of the single agent in DiFi cells and did not sensitize human colorectal tumor cells that were insensitive to the treatment with either single agent. Our results, presented here, together with a recent study reporting the antagonistic effect of cetuximab in combination with gefitinib in epidermoid carcinoma cell lines (27), underlie the need for further investigation on the therapeutic gains versus potential disadvantages (such as increased side effects, development of resistance) of combining anti-EGFR mAbs with the EGFR TKIs.

Grant support: Guildford Undetected Tumour Screening charity, Guildford, United Kingdom.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We are grateful to AstraZeneca for provision of the EGFR tyrosine kinase inhibitor gefitinib, and the GUTS charity (UK) for supporting this work.

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