Antitumor effect of afatinib, as a human epidermal growth factor receptor 2-targeted therapy, in lung cancers harboring HER2 oncogene alterations

Abstract

Human epidermal growth factor receptor 2 (HER2) is a member of the HER family of proteins containing four receptor tyrosine kinases. It plays an important role in the pathogenesis of certain human cancers. In non-small-cell lung cancer (NSCLC), HER2 amplification or mutations have been reported. However, little is known about the benefit of HER2-targeted therapy for NSCLCs harboring HER2 alterations. In this study, we investigated the antitumor effect of afatinib, an irreversible epidermal growth factor receptor (EGFR)-HER2 dual inhibitor, in lung cancers harboring HER2 oncogene alterations, including novel HER2 mutations in the transmembrane domain, which we recently identified. Normal bronchial epithelial cells, BEAS-2B, ectopically overexpressing wild-type HER2 or mutants (A775insYVMA, G776VC, G776LC, P780insGSP, V659E, and G660D) showed constitutive autophosphorylation of HER2 and activation of downstream signaling. They were sensitive to afatinib, but insensitive to gefitinib. Furthermore, we examined the antitumor activity of afatinib and gefitinib in several NSCLC cell lines, and investigated the association between their genetic alterations and sensitivity to afatinib treatment. In HER2-altered NSCLC cells (H2170, Calu-3, and H1781), afatinib downregulated the phosphorylation of HER2 and EGFR as well as their downstream signaling, and induced an antiproliferative effect through G1 arrest and apoptotic cell death. In contrast, HER2- or EGFR-non-dependent NSCLC cells were insensitive to afatinib. In addition, these effects were confirmed in vivo by using a xenograft mouse model of HER2-altered lung cancer cells. Our results suggest that afatinib is a therapeutic option as a HER2-targeted therapy for NSCLC harboring HER2 amplification or mutations.

Keywords: Afatinib; ERBB2; HER2; HER2-targeted therapy; non-small-cell lung cancer.

Figure 1

Overexpressing wild‐type or mutant HER2 activates human epidermal growth factor receptor 2 (HER2) signaling, and afatinib inhibits them. (a) BEAS‐2B cells were transiently transfected with wild‐type HER2, its variants, A775insYVMA, G776VC, G776LC, P780insGSP, G660D, and V659E, or vector control. Approximately 36 h after the transfection, cells were serum‐starved overnight. Lysates were subjected to Western blot analysis with the indicated antibodies. (b) Location of the major mutation site and the ATP‐binding pocket of the HER2 kinase domain in the modeled HER2–afatinib complex structure. Afatinib is shown as a stick. Residues at the mutation site of the HER2 kinase domains (A775, G776, and P780) are shown as spheres. These residues are concentrated in an exposed area on the protein surface (A775–P780) of the αC–β4 loop (M774–R784), which is located on the back side of the ATP‐binding pocket. (c) Forty‐eight hours after the transfection, cells were treated with or without 1.0 μM gefitinib or 0.1 μM afatinib for 6 h. Lysates were subjected to Western blot analysis with the indicated antibodies. EGFR, epidermal growth factor receptor.

Figure 2

Afatinib inhibits both HER2‐amplified and HER2‐mutant non‐small‐cell lung cancer and breast cancer cells. (a) HER2‐amplified and HER2‐mutant non‐small‐cell lung cancer cells were treated with afatinib or gefitinib for 72 h and IC ₅₀ values were determined using an MTS assay. Error bars indicate standard deviations. (b) Cells were treated with the indicated concentrations of afatinib for 6 h and lysates were subjected to Western blot analysis with the indicated antibodies. EGFR, epidermal growth factor receptor.

Figure 3

Afatinib induces cell cycle arrest and apoptosis in HER2‐dependent lung cancer cells. (a) Non‐small‐cell lung cancer cells were treated with 0.1 μM afatinib for 24 or 48 h and subjected to cell cycle analysis using flow cytometry. The graph represents the mean percentages of each phase in live cells ± SD of triplicate cultures. *P < 0.05 versus vehicle controls by one‐way anova followed by Dunnett's test. (b) Lysates from non‐small‐cell lung cancer cells were collected at the indicated time points after the addition of 0.1 μM afatinib and subjected to Western blot analysis with the indicated antibodies. PARP, poly(ADP‐ribose) polymerase.

Figure 4

Afatinib shows a strong antitumor effect on tumor growth in xenograft mouse model of HER2‐altered lung cancer cells. (a) Mice with H2170 tumors were given vehicle or afatinib. Tumor volume was determined at the indicated days after the onset of treatment. Data represent mean ± SE (n = 8). *P < 0.05 versus vehicle controls by two‐way anova for repeated measurements. (b) Appearance of H2170 tumors after treatment at the time the mice were killed. (c) Mice with H1781 tumors were given vehicle or afatinib and analyzed as in (a). Data represent mean ± SE (n = 8). *P < 0.05 versus vehicle controls by two‐way anova for repeated measurements. (d) Appearance of H1781 tumors after treatment at the time the mice were killed.