Pyrotinib targeted EGFR/GRP78 mediated cell apoptosis in high EGFR gene copy number gastric cancer

Despite frequent Epidermal Growth Factor Receptor (EGFR) amplification and overexpression in gastric cancer, limited therapeutic responses were observed in existing EGFR-targeted agents. Pyrotinib, an irreversible dual EGFR/HER2 tyrosine kinase inhibitor, has shown clinical efficacy in HER2-driven malignancies, but its potential role in EGFR-high copy number gastric cancer remains to be investigated.

Using EGFR-high copy number gastric cancer cell lines, primary cells and subcutaneous tumor models in nude mice, we systematically evaluated pyrotinib's anti-tumor activity through viability assays, apoptosis analysis, and transcriptomic profiling. Mechanistic studies included co-immunoprecipitation, proximity ligation assays, ubiquitination assays, and RNA sequencing.

Pyrotinib selectively suppressed proliferation, induced apoptosis, and chemosensitized in EGFR-high copy number gastric cancer models. Mechanistically, pyrotinib promoted EGFR-GRP78 (Glucose-regulated protein 78) complex formation in the endoplasmic reticulum, activating the protein kinase R-like endoplasmic reticulum kinase/ activating transcription factor 4/ C-EBP homologous protein (PERK/ATF4/CHOP) axis to drive ER stress-mediated apoptosis. Concurrently, pyrotinib inhibited GRP78 phosphorylation at Thr62, triggering K48-linked ubiquitination (ubiquitin chains formed via lysine 48 linkages) and proteasomal degradation, which impaired DNA double-strand break (DSB) repair and sensitized cells to oxaliplatin-induced γ-H2A.X accumulation.

This translational study suggests that pyrotinib combined with oxaliplatin may serve as a promising strategy for patients with EGFR-high copy number gastric cancer and highlighted the discovery of this previously unknown EGFR/ GRP78 signaling axis, which provides the molecular basis and the rationale to target EGFR.