ERBB3 (HER3) is a key sensor in the regulation of ERBB-mediated signaling in both low and high ERBB2 (HER2) expressing cancer cells

Abstract

Aberrant expression and activation of EGFR and ERBB2 (HER2) have been successfully targeted for cancer therapeutics. Recent evidence from both basic and clinical studies suggests that ERBB3 (HER3) serves as a key activator of downstream signaling through dimerization with other ERBB proteins and plays a critical role in the widespread clinical resistance to EGFR and HER2 targeting cancer therapies. As a result, HER3 is actively pursued as an antibody therapeutic target for cancer. Ligand binding is thought to be a prerequisite for dimerization of HER3 with other ERBB proteins, which results in phosphorylation of its c-terminal tyrosine residues and activation of downstream AKT and MAPK signaling pathways. In this study, we report that an anti-HER2 monoclonal antibody (HER2Mab), which blocks HER2 dimerization with HER3, induces HER3 dimerization with EGFR in both low and high HER2 expressing cancer cells. Treatment of the low HER2 expressing MCF7 cancer cells with HER2Mab promoted cell proliferation and migration in the absence of HER3 ligand stimulation. Follow-up studies revealed that HER2Mab-induced HER3 signaling via EGFR/HER3 dimerization and activation of downstream AKT signaling pathways. These results suggest that equilibrium of dimerization among the ERBB proteins can be perturbed by HER2Mab and HER3 plays a key role in sensing the perturbation.

Keywords: Anti-HER2 antibody; EGFR; ERBB2/HER2; ERBB3/HER3; MCF7; signaling.

Figure 1

HER2Mab-induced cell proliferation of MCF7 cells in the absence of NRG1. MCF7 and MCF7-HER2 cells were trypsinized and seeded at 5000 cells/well in low serum (1%) medium in an E-plate (Roche). Cells were allowed for attachment to the plate for 3 h. HER2Mab (10 μg/mL) and/or NRG1 (100 ng/mL) were added according to the study design. Arrows indicated starting point of treatment. Cell proliferation was monitored label-free for 144 h at 37°C with an xCELLigence instrument (Roche). Both cell growth index curves and cell growth as percent of control at 144 h were presented. Each treatment contains eight replications and standard errors are shown on the bar graphs. Student's t-test was applied for pairwise statistical comparisons, *P < 0.05; **P < 0.01; ***P < 0.001. (a) MCF7 cells treated with HER2Mab; (b) MCF7 cells treated with NRG-1 and HER2Mab/NRG1; (c) MCF7-HER2 cells treated with HER2Mab; and (d) MCF7-HER2 cells treated with NRG1 and HER2Mab/NRG1.

Figure 2

HER2Mab-induced cell migration of MCF7 cells in the absence of NRG1. Trypsinized MCF7 (a) or MCF7-HER2 (b) cells (1 × 10⁶) were seeded into Transwell plate (Costar, Corning, NY), and treated with PBS (c), NRG1 (100 ng/mL) (N), 10 μg/mL of HER2Mab (H), or combined NRG1 and HER2Mab treatment (NH). After 18 h incubation at 37°C, migrated cells were stained with 0.5% crystal violet for 5 min and visualized under a Carl Zeiss fluorescence microscope. Each treatment contains eight replications and standard errors are shown on the bar graph. Student's t-test was applied for pairwise statistical comparisons, *P < 0.05; **P < 0.01; ***P < 0.001. C, basal migration; N, cells treated with NRG1; H, cells treated with HER2Mab; NH, cells under the combined treatment of NRG1 and HER2Mab.

Figure 3

HER2Mab-induced ligand-independent EGFR/HER3 dimerization in MCF7 cells. HER3/EGFR or HER3/HER2 dimerization was visualized by the proximity ligation assay (PLA) protocol [11]. After serum starved with low serum media (1%) for 24 h, MCF7 cells were treated with PBS (C), NRG1 (N), HER2Mab (H), or combined HER2Mab/NRG1 (NH). After performing the PLA procedure, images were taken with a Carl Zeiss Fluorescence microscope, red spots which represent individual dimers from 10 randomly selected images were counted and plotted. Student's t-test was applied for pairwise statistical comparisons between treatments, *P < 0.05; **P < 0.01; ***P < 0.001. (a) A representative image of basal EGFR/HER3 dimerization in MCF7 cells. (b) EGFR/HER3 dimerization in MCF7 cells treated with NRG1. (c) EGFR/HER3 dimerization in MCF7 cells treated with HER2Mab. (d) EGFR/HER3 dimerization in MCF7 cells treated with HER2Mab and NRG1. (e) Statistical analysis of treatments (a–d). (f) Basal HER2/HER3 interaction in MCF7 cells. (g) HER2/HER3 interaction in MCF7 cells treated with NRG1. (h) HER2/HER3 interaction in MCF7 cells treated with HER2Mab. (i) HER2/HER3 interaction in MCF7 cells treated with HER2Mab and NRG1. (j) Statistical analysis of treatments (f–i).

Figure 4

HER2Mab effectively blocked HER2/HER3 dimerization in MCF7-HER2 cells. HER3/EGFR or HER3/HER2 dimerization was visualized by the proximity ligation assay (PLA) protocol [11]. After serum starved with low serum media (1%) for 24 h, MCF7-HER2 cells were treated with NRG1 (N), HER2Mab (H), or combined HER2Mab/NRG1 (NH). After performing the PLA procedure, images were taken with a Carl Zeiss Fluorescence microscope, red spots which represent individual dimers from 10 randomly selected images were counted and plotted. Student's t-test was applied for pairwise statistical comparisons between treatments, *P < 0.05; **P < 0.01; ***P < 0.001. (a) A representative image of basal EGFR/HER3 dimerization in MCF7-HER2 cells. (b) EGFR/HER3 dimerization in MCF7-HER2 cells treated with NRG-1. (c) EGFR/HER3 dimerization in MCF7-HER2 cells treated with HER2Mab. (d) EGFR/HER3 dimerization in MCF7-HER2 cells treated with HER2Mab and NRG1. (e) Statistical analysis of treatments (a–d). (f) Basal HER2/HER3 interaction in MCF7-HER2 cells. (g) HER2/HER3 interaction in MCF7-HER2 cells treated NRG1. (h) HER2/HER3 interaction in MCF7-HER2 cells treated with HER2Mab. (i) HER2/HER3 interaction in MCF7-HER2 cells treated with HER2Mab and NRG1. (j) Statistical analysis of treatments (f–i).

Figure 5

HER2Mab-induced ligand-independent HER3 phosphorylation and AKT phosphorylation in MCF7 and MCF7-HER2 cells. (a) p-HER3(Y1289) in MCF7 and MCF7-HER2 cells in response to NRG1 and HER2Mab treatment. Western gel images showed were quantitated using the ImageJ software after multiple images were taken from separate experiments (n = 3). (b) pAKT(S473) in MCF7 and MCF7-HER2 cells in response to NRG1 and HER2Mab treatment as determined by Western blot. (c) pAKT(S473) in MCF7 and MCF7-HER2 cells in response to NRG1 and HER2Mab treatment as determined using the nanofluidic immunoassay. Peaks in the boxed area indicate pAKT(S473). (d) pERK1/2 in MCF7 and MCF7-HER2 cells in response to NRG1 and HER2Mab treatment as determined by Western blot.