Anti-EGFR monoclonal antibodies and EGFR tyrosine kinase inhibitors as combination therapy for triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is characterized by overexpression of epidermal growth factor receptor (EGFR) and activation of its downstream signaling pathways. Dual targeting of EGFR using one monoclonal antibody (mAb; cetuximab or panitumumab) and one tyrosine kinase inhibitor (EGFR-TKI; gefitinib or erlotinib) is a potential therapeutic approach. We investigated the effect of these therapies in EGFR-expressing TNBC cell lines that do or do not harbor the main activating mutations of EGFR pathways. Cell lines were sensitive to EGFR-TKIs, whereas mAbs were active only in MDA-MB-468 (EGFR amplification) and SUM-1315 (KRAS and PTEN wild-type) cells. MDA-MB-231 (KRAS mutated) and HCC-1937 (PTEN deletion) cells were resistant to mAbs. The combined treatment resulted in a synergistic effect on cell proliferation and superior inhibition of the RAS/MAPK signaling pathway in mAb-sensitive cells. The anti-proliferative effect was associated with G1 cell cycle arrest followed by apoptosis. Sensitivity to therapies was characterized by induction of positive regulators and inactivation of negative regulators of cell cycle. These results suggest that dual EGFR inhibition might result in an enhanced antitumor effect in a subgroup of TNBC. The status of EGFR, KRAS and PTEN could be used as a molecular marker for predicting the response to this therapeutic strategy.

Keywords: anti-EGFR targeted therapy; cell cycle; cytotoxicity; epidermal growth factor receptor; triple-negative breast cancer.

Figure 1. Overexpression of phosphorylated EGFR, AKT and ERK1/2 in TNBC cell lines

Basal levels of EGFR, AKT, ERK1/2 and their phosphorylated forms were evaluated by Western blot analysis in TNBC cell lines and the non-TNBC MCF-7 cell line after 24 h of culture. Cells were lysed, and 15 μg of whole cell protein extract was separated by 10% SDS-PAGE and immunoblotted using the indicated antibodies. Beta-actin antibody was used as a loading control. The data shown are representative of two independent experiments. Bar charts depict densitometric quantification of Western blot signals as described in the Materials and Methods.

Figure 2. Viability assay of TNBC cell lines treated with anti-EGFR-targeted therapies

A. Cells were treated for 24 h with increasing concentrations of cetuximab or panitumumab and B. with the indicated concentrations of EGFR-TKIs (erlotinib (ERLO) or gefitinib (GEF)) combined with 10 μg/ml anti-EGFR mAbs (cetuximab (CX) or panitumumab (PN)). Cell viability was assessed using the SRB assay as described in the Materials and Methods. The results are expressed as percent of viability of untreated cells and are the mean values ± SEM of three independent experiments. Dashed curves represent the expected Bliss values if the combined effects were additive. The theoretical Bliss curves are shown for mAb-sensitive cell lines (MDA-MB-468 and SUM-1315). *p<0.05 for comparison using two-way analysis of variance (ANOVA) followed by an unpaired Student's t-test between cells treated with EGFR-TKIs and cells treated with combination therapy.

Figure 3. Western blot analysis of EGFR, AKT, ERK 1/2 and their phosphorylated forms in TNBC cell lines treated with a combination of anti-EGFR-targeted therapies

Cell lines were exposed to 5 μM of erlotinib (ERLO) or gefitinib (GEF) and 10 μg/ml of cetuximab (CX) or panitumumab (PN) for 24 h. Fifty μg of whole cell protein extract was analyzed via 10% SDS-PAGE and immunoblotted using the indicated antibodies. Beta-actin antibody was used as a loading control. The data shown are representative of two independent experiments.

Figure 4. Effect of anti-EGFR-targeted therapies on cell cycle progression in TNBC cell lines

Cells were treated for 48 h with 10 μg/ml of cetuximab (CX) or panitumumab (PN) and 5 μM of erlotinib (ERLO) or gefitinib (GEF) and stained with propidium iodide. Fluorescence was analyzed for cell cycle distribution by flow cytometry. The results are the mean values of three independent experiments. *p<0.05 for comparison between cells treated and cells untreated (CLTR) using Student's t-test.

Figure 5. Apoptotic effect of anti-EGFR-targeted therapies in TNBC cell lines

Cells were treated for 48 h with 10 μg/ml of cetuximab (CX) or panitumumab (PN) and 5 μM of erlotinib (ERLO) or gefitinib (GEF). Analysis of apoptosis was performed by both Annexin V-FITC and propidium iodide staining. Quantification of positive cells was evaluated by flow cytometry, and apoptotic cells were expressed as a percentage of total cell number. The data represent the mean values ± SEM of triplicate experiments. *p<0.05 for comparison between treated cells and untreated cells (CLTR) using Student's t-test.

Figure 6. Profiles of differentially expressed genes in TNBC cell lines used in this study

The data are presented in heat map format combined to hierarchical clustering. Each row represents a gene, and each column represents a cell line and treatments. The expression of each gene is relative to the mean gene expression in all cell lines and is illustrated according to a color scale from green to red. Genes in red and green indicate expression above and below the median, respectively. Cell lines were exposed for 48 h to 5 μM of erlotinib (erlo) or gefitinib (gef) and/or 10 μg/ml of cetuximab (cx) or panitumumab (pn). Two independent experiments were performed; 1-1′: untreated cells; 2-2′: cx; 3-3′: pn; 4-4′: erlo; 5-5′: erlo + cx; 6-6′: erlo + pn; 7-7′: gef; 8-8′: gef + cx; 9-9′: gef + pn.

Figure 7. A suggested algorithm for predicting the response to treatment with a combination of dual EGFR inhibitory agents according to EGFR, KRAS and PTEN status

mAbs: monoclonal antibodies; EGFR-TKIs: tyrosine kinase inhibitors; wt: wild-type; mut: mutated; amp: amplification.