Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

Abstract

Anti-HER2 therapy is integral to the treatment of HER2-positive breast cancer, but drug resistance hampers its effectiveness. Although antibody-drug conjugates (ADCs) are increasingly used in clinical practice, their application is often hindered by adverse reactions and drug resistance. Therefore, it is crucial to enhance the bioavailability of ADCs and reduce their dosages to mitigate both adverse effects and resistance. Pyrotinib's effect on HER2-positive breast cancer cell lines (SK-BR-3 and JIMT-1) was investigated via western blot, focusing on HER2 and downstream pathways. Pyrotinib's influence on HER2 ubiquitination and internalization was assessed through RT-qPCR, western blot, and immunofluorescence. The ability of pyrotinib to augment trastuzumab emtansine (T-DM1) endocytosis and antiproliferative effects was studied via CCK-8 and immunofluorescence. In vivo experiments in nude mice were conducted to explore the therapeutic efficacy of T-DM1 combined with pyrotinib. The single-drug study showed that pyrotinib downregulated HER2 protein levels and HER2 downstream signaling pathways. The mechanism of downregulating HER2 protein levels involved the promotion of HER2 internalization and degradation through the ubiquitin-proteasome pathway. The two-drug combination study showed that pyrotinib promoted the endocytosis of T-DM1, which improved its bioavailability. Increased cellular uptake further enhanced the antitumor effects of T-DM1 in both in vitro and in vivo experiments. Our results reveal the molecular mechanism by which pyrotinib regulates HER2 levels by promoting HER2 internalization, thereby facilitating the endocytosis of T-DM1. These findings suggest a potential combination treatment strategy for the targeted therapy of HER2-positive breast cancer.

Keywords: Breast cancer; Endocytoses; Human epidermal growth factor receptor 2; Trastuzumab emtansine; Tyrosine kinase inhibitor.

Fig. 1

Pyrotinib downregulates HER2 protein levels and suppresses phosphorylation of HER2, PI3K/AKT, and RAS/MAPK signaling pathways in SK-BR-3 and JIMT-1 cells. (a–d) Expression levels of HER2 and its downstream proteins in the PI3K/AKT and RAS/MAPK signaling pathways were analyzed by western blotting. (a) Both cell types were treated with lapatinib (0.001, 0.01, 0.1, 1 µM) for 24 h. (b) Both cell types were treated with lapatinib (1 µM) at different time points. (c) Both cell types were treated with pyrotinib (0.001, 0.01, 0.1, 1 µM) for 24 h. (d) Both cell types were treated with pyrotinib (0.1 µM) at different time points. Results are representative of 3 independent replicates.

Fig. 2

Pyrotinib promotes HER2 degradation via the ubiquitin–proteasome pathway. (a) HER2 mRNA expression in SK-BR-3 and JIMT-1 cells treated with pyrotinib (0.5µM) or lapatinib (1µM) as assessed using RT-qPCR. (b–d) SK-BR-3 and JIMT-1 cells were treated with the lysosomal inhibitor Baf-A1 (20nM) or proteasome inhibitors Velcade (0.5 µM) or MG-132 (10µM) for 0.5 h, with DMSO as the control, followed by the addition of pyrotinib (0.5µM) for 0, 2, and 4 h. (e–f) Cells were subjected to MG132 (10 µM) treatment for 0.5 h or DMSO as the control, followed by the addition of pyrotinib (0.5 µM) for 0, 2, and 4 h. HER2 was immunoprecipitated from the lysates, and the samples were analyzed by immunoblotting with an anti-ubiquitin, anti-HER2, and anti-HSP70 antibodies. GAPDH served as the loading control. Results are representative of 3 independent replicates.

Fig. 3

Pyrotinib promotes HER2 internalization and T-DM1 endocytosis. (a) Cells were treated with pyrotinib (0.5µM) or lapatinib (1 µM) for 0, 2, and 4 h and processed for immunofluorescence experiments using anti-HER2 antibody (green). Nuclei were stained with DAPI (blue) (×1000), Scale bar = 10 μm. (b) After labeling T-DM1 with pHrodo Deep Red (pHro-do-T-DM1), the cells were exposed to pHrodo-T-DM1 (1 µg/mL) alone or in combination with pyrotinib (0.1µM) for 0, 2, and 4 h. pHrodo-T-DM1 emits red fluorescent signals within the cellular interior. Nuclei were stained with DAPI (blue) (×600). The quantification of T-DM1 fluorescence intensity is now shown in Supplementary Figure S2. Scale bar = 10 μm. Results are representative of 3 independent replicates.

Fig. 4

Pyrotinib enhances the antitumor effect of T-DM1 in vitro. (a) The effects of various concentrations of pyrotinib and lapatinib on the viability of SK-BR-3 and JIMT-1 cells. Cell viability was assessed using a CCK-8 assay after 24 h. Y-axis: The Inhibition Ratio (%) represents the percentage of cell proliferation inhibition compared to untreated/control cells. (b) Cells were treated with T-DM1 (0.0001, 0.001, 0.01, 0.1, 1, 10 µg/mL) either alone or in combination with pyrotinib or lapatinib (SK-BR-3:0.01µM, JIMT-1:0.05µM). Cell viability was measured after 24 h of treatment. (c) Scratch assay to detect the effect of pyrotinib or lapatinib(SK-BR-3:0.01µM, JIMT-1:0.05µM) combined with T-DM1 (0.1 µg/mL) on the migration ability of breast cancer cells. Photograph the scratched area after 24 h. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Results are representative of 3 independent replicates.

Fig. 5

Pyrotinib enhances the antitumor effect of T-DM1 in vivo. (a) Images of JIMT-1 xenografts harvested after 21 days of treatment with T-DM1 (10 mg/kg) with or without pyrotinib (2 mg/kg) (n = 6). (b) Changes in tumor weight in the examined mice. (c) Changes in tumor volume in the examined mice. (d) Body weight changes in mice after treatments. (e) Representative images displaying HE and IHC staining of xenograft tumor tissues (×400), scale bars = 50 μm. The histogram shows the average absorbance of HER2. (f) The protein expression levels of HER2 and its downstream signaling pathways in tumor tissues of each group were analyzed by western blot. GAPDH served as the loading control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Results are representative of 3 independent replicates.