A Novel Mechanism Underlying the Inhibitory Effects of Trastuzumab on the Growth of HER2-Positive Breast Cancer Cells

Abstract

To improve the efficacy of trastuzumab, it is essential to understand its mechanism of action. One of the significant issues that makes it difficult to determine the precise mechanism of trastuzumab action is the formation of various HER receptor dimers in HER2-positive breast cancer cells. So far, studies have focused on the role of HER2-HER3 heterodimers, and little is known regarding EGFR-HER2 heterodimers. Here, we study the role of trastuzumab on the cell signaling and cell proliferation mediated by EGFR-HER2 heterodimers in BT474 and SRBR3 cells. EGF stimulates the formation of both EGFR homodimer and EGFR-HER2 heterodimer. Trastuzumab only binds to HER2, not EGFR. Therefore, any effects of trastuzumab on EGF-induced activation of EGFR, HER2, and downstream signaling proteins, as well as cell proliferation, are through its effects on EGFR-HER2 heterodimers. We show that trastuzumab inhibits EGF-induced cell proliferation and cell cycle progression in BT474 and SKBR3 cells. Interestingly trastuzumab strongly inhibits EGF-induced Akt phosphorylation and slightly inhibits EGF-induced Erk activation, in both BT474 and SKBR3 cells. These data suggest the presence of a novel mechanism that allows trastuzumab to inhibit EGR-induced Akt activation and cell proliferation, without blocking EGF-induced EGFR-HER2 heterodimerization and activation. We show that trastuzumab inhibits EGF-induced lipid raft localization of the EGFR-HER2 heterodimer. Disruption of the lipid raft with MβCD blocks HER2-mediated AKT activation in a similar way to trastuzumab. MβCD and trastuzumab synergically inhibit AKT activation. We conclude that trastuzumab inhibits EGF-induced lipid raft localization of EGFR-HER2 heterodimer, which leads to the inhibition of Akt phosphorylation and cell proliferation, without blocking the formation and phosphorylation of the EGFR-HER2 heterodimer.

Keywords: EGF; EGFR; HER2; akt; cell cycle progression; cell proliferation; dimerization; erk; lipid raft; phosphorylation; trastuzumab.

Figure 1

The effects of trastuzumab (TRZ) on the viability of BT474 and SKBR3 breast cancer cells in absence and presence of 10% FBS, and EGF. Cells were treated with 10 µg/mL concentration of trastuzumab for 72 h. Cells treated with normal human IgG were used as negative control and cells treated with CP–714724 (CP) at 1 µM concentration were used as positive control. Each value is the average of at least three experiments and the error bar is standard error. **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 2

The effects of trastuzumab (TRZ) on the cell cycle progression of BT474 (A) and SKBR3 (B) cells in presence and absence of 10% FBS. Cells were treated with 10 µg/mL concentration of trastuzumab for 72 h. Cells treated with normal human IgG were used as negative control and cells treated with CP-714724 (CP) at 1 µM concentration were used as positive control. Each value is the average of at least three experiments and the error bar is standard error. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 3

The effect of trastuzumab on HER2 heterodimer formation. The effect of trastuzumab at concentration of 10 µg/mL on HER2–EGFR and HER2–HER3 heterodimers in BT474 (A) and SKBR3 (B) breast cancer cells for 1 h. HER2–EGFR and HER2–HER3 heterodimer formation was assessed in the presence and absence of EGF and HRGα at 50 ng/mL, respectively. The expression of HER receptors in both IP and total cell lysate (TCL) samples was revealed by immunoblotting. HER2 receptors were precipitated using HER2-specific antibody as primary antibody, followed by immunoblotting with the indicated antibodies. Cells treated with normal human IgG were used as negative control. **: p < 0.01.

Figure 4

The effects of trastuzumab on the phosphorylation of HER receptors in BT474 (A) and SKBR3 (B) cells. The cells treated with trastuzumab at concentration of 10 µg/mL in the presence and absence of EGF at 50 ng/mL concentration. The phosphorylation of HER receptors was revealed by immunoblotting. Cells treated with normal human IgG were used as negative control and cells treated with CP–714724 (CP) at 1 µM concentration were used as positive control. Each value is the average of at least three experiments and the error bar is standard error. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 5

The effects of trastuzumab on EGF-induced phosphorylation of EGFR and HER2 in BT474 and SKBR3 cells. Cells were incubated with TRZ (10 µg/mL) or human IgG for 1 h and then stimulated with EGF (50 ng/mL). (A) Following EGF stimulation for 15 min, the phosphorylation of EGFR and HER2 (green) was determined by antibody to phosphorylated EGFR (pEGFR, pY1086) and pHER2 (pY1139), respectively. The localization of EGFR and HER2 (red) was determined by antibodies to by total EGFR and HER2, respectively. Yellow indicates the co–localization. the size bar = 20 µm. (B) Localization of pEGFR (pY1086) and pHER2 (pY1139) following EGF stimulation for 30 min. the size bar = 20 µm.

Figure 6

The effects of trastuzumab on EGF-induced phosphorylation of HER2 at various pY sites including pY1005, pY1112, pY1127, pY1196, and pY1248 in BT474 and SKBR3 cells. Cells were incubated with TRZ (10 µg/mL) or human IgG for 1 h and then stimulated with EGF (50 ng/mL) for 15 min. The phosphorylation of various HER2 pY sites (green) were determined by specific antibodies followed by FITC conjugated secondary antibody, and total HER2 (red) was revealed by anti-HER2 antibody followed by TRITC conjugated secondary antibody. Yellow indicates the co-localization. the size bar = 20 µm.

Figure 7

The effects of trastuzumab on HER2-mediated downstream signaling pathway in BT474 and SKBR3 cells. The cells were treated with trastuzumab at concentration of 10 µg/mL in the presence and absence of EGF (50 ng/mL). The phosphorylation of Akt at threonine (T) 308 and serine (S) 473 phosphorylation sites as well as Erk phosphorylation were revealed by immunoblotting. Cells treated with normal human IgG were used as negative control and cells treated with CP–714724 (CP) at 1 µM were used as positive control. Each value is the average of at least three experiments and the error bar is standard error. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.

Figure 8

The effect of trastuzumab on EGF-induced lipid raft localization of EGFR–HER2 heterodimer in BT474 cells. (A) EGFR and HER2 level in isolated lipid rafts. The cells were incubated with trastuzumab or normal human IgG (10 µg/mL) for 1 h and then stimulated with EGF (50 ng/mL) for 30 min. Proteins associated with lipid rafts were isolated by using Bio-Rad ReadyPrep™ Protein Extraction Kit (Signal). The level of HER2 and EGFR receptors in isolated lipid rafts were examined by immunoblotting. Each value is the average of three experiments and the error bar is the standard error. *: p < 0.05, **: p < 0.01. (B) Co-localization (yellow) of pEGFR (red) and pHER2 (red) with Alex Fluor 488 conjugated CT-B (green). Cells were incubated with CB-T (10 µg/mL) with or without trastuzumab (10 µg/mL) for 1 h and then stimulated with EGF for 30 min. The localization of EGFR or HER2 was revealed by TRITC conjugated secondary antibody following the incubation with the primary antibody. Nucleus was counter stained with Dapi.

Figure 9

Disruption of lipid raft by MβCD and the effects on EGF-induced Akt phosphorylation. BT474 cells were treated with MβCD at various concentrations for 1 h to disrupt lipid raft. For the experiments with trastuzumab treatment, cells were incubated with trastuzumab and MβCD together for 1 h. Cells were then stimulated with EGF for 30 min. (A) CT-B stain (green) to show the disruption of lipid raft by MβCD. Cells were also stained with pHER2 (red). Line scan showed the intensities of CT-B and pHER2. (B) Dose-dependent effects of MβCD on Akt phosphorylation in BT474 breast cancer cells. Cells were treated with MβCD at indicated concentration for 4 h. The phosphorylation of Akt was then examined by immunoblotting. (C) Synergistic effects of trastuzumab and MβCD on Akt phosphorylation. Cells were treated with MβCD alone at 10 and 20 mM concentrations, or in combination with trastuzumab at 10 μg/mL. The phosphorylation of Akt was then examined by immunoblotting. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001.