Amphiregulin regulates the activation of ERK and Akt through epidermal growth factor receptor and HER3 signals involved in the progression of pancreatic cancer

Abstract

Pancreatic cancer is one of the most lethal malignancies. Epidermal growth factor receptor (EGFR), HER3, Akt, and amphiregulin have been recognized as targets for pancreatic cancer therapy. Although gemcitabine + erlotinib has been the recommended chemotherapy for pancreatic cancer, the prognosis is extremely poor. The development of molecularly targeted therapies has been required for patients with pancreatic cancer. To assess the validation of amphiregulin as a target for pancreatic cancer therapy, we examined its expression in pancreatic cancer using real-time PCR analyses and ELISA. We also measured the apoptotic cell rate using TUNEL assays. In addition, alterations in signaling pathways were detected by immunoblotting analyses. Treatment with gemcitabine, which reduced the cell viability and augmented the cell apoptotic rate, activated and subsequently attenuated ERK and EGFR signals. However, gemcitabine, paclitaxel, or cisplatin treatment enhanced the Akt activation, heterodimer formation of EGFR with HER3, and secretion of amphiregulin, indicating that the presence of gemcitabine promoted the activity of targeted molecules including amphiregulin, Akt, and HER3 for pancreatic cancer therapy. Combined treatment with an inhibitor for amphiregulin and gemcitabine, paclitaxel, or cisplatin induced synergistic antitumor effects, accompanied by the suppression of Akt and ERK activation. Blockade of amphiregulin suppressed the activities of EGFR, HER3, and Akt and the expression of amphiregulin itself. According to this evidence, combination chemotherapy of conventional anticancer drugs plus an inhibitor for amphiregulin would allow us to provide more favorable clinical outcomes for patients with pancreatic cancer.

Figure 1

Cell characteristics in pancreatic cancer. Differences in the expressions of epidermal growth factor receptor (EGFR) ligands in pancreatic cancer patients (a) and pancreatic cancer cell lines (b). Each value represents the mean and SD of the mRNA expression index for an EGFR ligand (n = 4). Closed blue circles HB‐EGF; closed green circles, epiregulin; closed red circles, amphiregulin; closed yellow circles, transforming growth factor (TGF)‐α; open blue circles, betacellulin; open green circles, epigen; open red circles, EGF. *P < 0.05 versus each value of the other six EGFR ligands; **P < 0.05 versus each value of the other five EGFR ligands. (c) Amounts of EGFR ligands in culture media from cancer cells incubated for 48 h. The concentrations of HB‐EGF, amphiregulin, TGF‐α, and EGF are presented as the concentrations per 1 × 10⁶ cells. Blue bars, TGF‐α; red bars, amphiregulin; yellow bars, HB‐EGF; EGF could not be determined. Each value represents the mean and SD (n = 3). *P < 0.05 versus each amount of HB‐EGF, TGF‐α, or EGF. (d) Differences in the protein expressions of EGFR, HER2, HER3, HER4, Akt, ERK, and β‐actin in pancreatic cell lines.

Figure 2

Cell behavior after treatment with gemcitabine in AsPC‐1 pancreatic cancer cells. Differences in the cell apoptotic rate (a), cell viability rate (b), and amount of amphiregulin in the culture medium (c) after treatment with various doses of gemcitabine for 48 h. Each value represents the mean and SD (n = 4). *P < 0.05 versus the value of the cell apoptotic rate, cell viability rate, or concentration of amphiregulin without gemcitabine treatment. (d) Alterations in the expressions of phosphorylated epidermal growth factor receptor (EGFR), HER3, Akt, and ERK after treatment with various doses of gemcitabine for 48 h. (e) Analysis of the heterodimer formation of EGFR with HER3 in the presence of various doses of gemcitabine. ip, immunoprecipitation; WB, western blotting.

Figure 3

Antitumor effects of combined treatment with gemcitabine and a variety of inhibitors in AsPC‐1 pancreatic cancer cells. Alterations in the expression of phosphorylated Akt (a) and ERK (b) after combined treatment with gemcitabine and an inhibitory anti‐amphiregulin antibody (10 μg/mL), cetuximab (10 μg/mL), erlotinib (1 μM), or an inhibitory anti‐HER3 antibody (10 μg/mL) for 48 h. Differences in the cell apoptotic rates (c) and amounts of amphiregulin in the culture medium (d) after treatment with gemcitabine (0.1 μM) with or without each inhibitory antibody against amphiregulin (10 μg/mL), transforming growth factor (TGF)‐α (10 μg/mL), neuregulin (10 μg/mL), or HER3 (10 μg/mL), cross‐reacting material (CRM)197 (10 μg/mL), cetuximab (10 μg/mL), or erlotinib (1 μM). Each value represents the mean and SD (n = 4). *P < 0.05 versus the value of the cell apoptotic rate after treatment with the inhibitory anti‐amphiregulin antibody with gemcitabine (0.1 μM); **P < 0.05 versus the value of the cell apoptotic rate after treatment with the inhibitory anti‐amphiregulin antibody without gemcitabine; ***P < 0.05 versus the value of the corresponding amount of amphiregulin only in the treatment with each inhibitor (minus 0.1 μM gemcitabine).

Figure 4

Activation of Akt and ERK mediated by heterodimer formation of epidermal growth factor receptor (EGFR) with HER3 in AsPC‐1 pancreatic cancer cells. (a) Phosphorylation of Akt (upper panels) and ERK (lower panels) stimulated by amphiregulin (50 ng/mL) or neuregulin (50 ng/mL) in the absence or presence of inhibitory anti‐amphiregulin (10 μg/mL), anti‐neuregulin (10 μg/mL), or anti‐HER3 (10 μg/mL) antibodies. (b) Stimulation of heterodimer formation of EGFR with HER3 by amphiregulin (50 ng/mL) or neuregulin (50 ng/mL) in the absence or presence of inhibitory anti‐amphiregulin (10 μg/mL), anti‐neuregulin (10 μg/mL), or anti‐HER3 (10 μg/mL) antibodies. ip, immunoprecipitation; WB, western blotting.

Figure 5

Antitumor effects of combined treatment with gemcitabine and a variety of inhibitors in AsPC‐1 cells using a Matrigel 3D culture system. (a) Appearances of growing cells by phase‐contrast microscopy after treatment with control IgG (10 μg/mL), an inhibitory anti‐amphiregulin antibody (10 μg/mL), or cetuximab (10 μg/mL) with or without gemcitabine (0.1 μM) for 48 h. Differences in the cell number (b), cell apoptotic rate (c), and concentration of amphiregulin in the culture medium (d) after treatment with control IgG (10 μg/mL), an inhibitory anti‐amphiregulin antibody (10 μg/mL), an inhibitory anti‐HER3 antibody (10 μg/mL), or cetuximab (10 μg/mL) with or without gemcitabine (0.1 μM) for 48 h. Each value represents the mean and SD (n = 4). *P < 0.05 versus the value for the cell number or apoptotic rate after treatment with an inhibitory anti‐amphiregulin antibody with gemcitabine treatment (0.1 μM); **P < 0.05 versus the value for the cell number or apoptotic rate after treatment with an inhibitory anti‐amphiregulin antibody without gemcitabine treatment.

Figure 6

Associations of therapeutic target molecules including amphiregulin (Amp), epidermal growth factor receptor (EGFR), HER3, and Akt with pancreatic cancer. (a) In pancreatic cancer, the abundant amount of amphiregulin enhances the activation of ERK through phosphorylation of EGFR and the activation of Akt through heterodimer formation of EGFR with HER3. (b) Treatment with gemcitabine (GEM) induces the dephosphorylation cell proliferative signals and stimulates marked secretion of amphiregulin, leading to formation of EGFR/HER3 heterodimer and further activation of Akt as a cell survival signal. (c) The combination of gemcitabine with an inhibitor for amphiregulin completely inhibits ERK and Akt activation. P, phosphorylation.