Inhibition of triple-negative breast cancer models by combinations of antibodies to EGFR

Abstract

Breast tumors lacking expression of human epidermal growth factor receptor 2 (HER2) and the estrogen and the progesterone receptors (triple negative; TNBC) are more aggressive than other disease subtypes, and no molecular targeted agents are currently available for their treatment. Because TNBC commonly displays EGF receptor (EGFR) expression, and combinations of monoclonal antibodies to EGFR effectively inhibit other tumor models, we addressed the relevance of this strategy to treatment of TNBC. Unlike a combination of the clinically approved monoclonal antibodies, cetuximab and panitumumab, which displaced each other and displayed no cooperative effects, several other combinations resulted in enhanced inhibition of TNBC's cell growth both in vitro and in animals. The ability of certain antibody mixtures to remove EGFR from the cell surface and to promote its intracellular degradation correlated with the inhibitory potential. However, unlike EGF-induced sorting of EGFR to lysosomal degradation, the antibody-induced pathway displayed independence from the intrinsic kinase activity and dimer formation ability of EGFR, and it largely avoided the recycling route. In conclusion, although TNBC clinical trials testing EGFR inhibitors reported lack of benefit, our results offer an alternative strategy that combines noncompetitive antibodies to achieve robust degradation of EGFR and tumor inhibition.

Fig. 1.

Specific combinations of anti-EGFR mAbs enhance receptor down-regulation and degradation. (A) MDA-MB-468 cells were incubated for 45 min at 4 °C with increasing concentrations of growth factors or mAbs. Thereafter, Texas Red-EGF (300 nM) was added for an additional 45 min. Fluorescence intensity was determined after washing using a microplate reader. The results represent averages (± SD) from three experiments. (B and C) Serum-starved MDA-MB-468 cells were incubated at 4 °C with the indicated mAbs (or with EGF). Cetuximab (B; ctx) or panitumumab (C; pan), each at 1 µg/mL, was added 15 min later and incubated for 45 min. The cells were then washed and incubated for 60 min at 4 °C with HRP-labeled anti human IgG. Light absorbance (415 nm) was determined using an ELISA microplate reader after washing and incubating for 15 min with 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). (D) HeLa cells were incubated for 12 h with the indicated mAbs (20 µg/mL) or for 60 min with EGF (10 ng/mL). Bound ligands were acid-stripped, and surface EGFR was measured by FACS analysis. Values are the average ± SD of triplicates. (E) HeLa cells were treated for 12 h with the indicated anti-EGFR mAbs (20 µg/mL) or for 60 min with EGF (10 ng/mL). Afterward, whole cell extracts were subjected to immunoblotting (IB) with the indicated antibodies. (F) HeLa cells were incubated for the indicated intervals with mAbs (20 µg/mL), or with EGF (10 ng/mL), and lysates were probed with the indicated antibodies.

Fig. 2.

Anti-EGFR antibodies enhance receptor ubiquitination and degradation. (A) Serum-starved HeLa cells were incubated with mAbs (10 µg/mL), a combination (each at 5 μg/mL), or with EGF (10 ng/mL), and lysates analyzed using immunoprecipitation (IP) and immunoblotting (IB). (B) HeLa cells transfected with a plasmid encoding an MYC peptide-tagged CBL, or an empty vector, were serum-starved and treated for 3 h with the indicated mAbs (20 µg/mL) or a combination (each at 10 µg/mL). Alternatively, cells were treated for 10 min with EGF (10 ng/mL). Lysates were probed as indicated. (C) HeLa cells that were preincubated (12 h) with bortezomib (2 µM) or bafilomycin (10 nM) were incubated (60 min) with EGF (10 ng/mL) or for 6 h with the indicated combination of mAbs (each at 10 µg/mL). Lysates were subjected to IB and signal quantification.

Fig. 3.

A combination of anti-EGFR antibodies enhances receptor internalization and impairs recycling. (A and B) Serum-starved HeLa cells were incubated with EGF (10 ng/mL; 30 min) or mAbs (20 µg/mL total; 4 h). Thereafter, cells were permeabilized and EGFR was localized using flow cytometry. EGFR distribution between the plasma membrane (scored as 1) and the cell’s center (scored as 0) was assessed. (C) HeLa cells were treated with EGF (10 ng/mL; 30 min) or mAbs (10 µg/mL total; 4 h). Following fixation, permeabilization, and incubation with anti-EGFR and anti-Rab11 antibodies, cells were incubated with fluorescent secondary antibodies, and images were acquired with a confocal fluorescent microscope. Colocalization of two markers appears as yellow (Merge).

Fig. 4.

A combination of anti-EGFR mAbs down-regulates a dimerization-defective mutant of EGFR but cannot trigger downstream signaling. (A) Serum-starved HeLa cells were incubated with EGF (10 ng/mL, 1 h) or with anti-EGFR mAbs (20 μg/mL total, 12 h) and lysates probed as indicated. (B) HeLa cells were transfected with a plasmid encoding a dimerization-defective mutant of EGFR (∆CR-EGFR-YFP). Forty-eight hours later, serum-starved cells were treated with EGF (10 ng/mL) or with mAbs (20 μg/mL total) before lysate immunoblotting. (C) HeLa cells were pretreated for 4 h with a selective EGFR kinase inhibitor (AG1478; 10 µM). Subsequently, cells were washed and treated with EGF (10 ng/mL), or with a combination of mAbs (each at 10 µg/mL). Lysates were probed with the indicated antibodies.

Fig. 5.

A combination of antibodies down-regulates EGFR and inhibits invasion of TNBC cells. (A) Whole extracts were prepared from the indicated cell lines after treatment with EGF (10 ng/mL, 1 h) or with the mAbs (20 µg/mL total, 6 h). Lysates were immunoblotted as indicated. (B) BT-549 cells were treated for 48 h with mAbs (20 μg/mL total) and lysates immunoblotted for EGFR and ERK2. (C and D) BT-549 cells were treated with mAbs as in (B). Thereafter, cells were plated in the upper compartment of invasion chambers. The lower compartments were filled with the respective mAb-containing media. Eighteen hours later, the filters were removed, fixed, permeabilized, and stained with methyl violet (0.3%). Cells growing on the upper side of the filter were removed and cells on the bottom side were photographed and quantified.

Fig. 6.

A combination of anti-EGFR mAbs interferes with EGF-dependent proliferation and with in vivo growth of TNBC cells. (A and B) HCC70 cells (3,000 cells per well) were seeded in 12-well plates and allowed to adhere overnight. Afterward, cells were incubated for 5 d in serum-free medium containing mAbs (20 µg/mL total) in the absence or presence of EGF (10 ng/mL). After fixation and staining with Giemsa (0.2% in saline), cells were photographed and quantified. Shown are the results of one of three experiments. (C and D) Groups of five CD1/nude mice were injected intradermally with 7 × 10⁶ HCC70 cells. Antibodies (total: 160 µg/animal/injection) were weekly injected intraperitoneally once tumors became palpable. Tumor volumes were assessed once per week. **P < 0.01 by two-way analysis of variance with Bonferroni’s multiple comparison posttests. The average tumor size in each group ± SEM is presented.