Fig. 4

Neratinib enhances the efficacy of CDK4/6 inhibitor combined with endocrine therapy in HR⁺/HER2-low breast cancer in vitro. (A,B) CCK8 and colony formation assays were performed to assess the proliferation of HR⁺/HER2-low breast cancer cells administered various concentrations of neratinib and CDK4/6 inhibitor, respectively, combined with endocrine therapy. For HER2-low cells: ET + 100 nM CDK4/6i + 125 nM neratinib, CI = 0.34; ET + 200 nM CDK4/6i + 250 nM neratinib, CI = 0.34; ET + 400 nM CDK4/6i + 500 nM neratinib, CI = 0.23; ET + 800 nM CDK4/6i + 1000 nM neratinib, CI = 0.46; ET + 1600 nM CDK4/6i + 2000 nM neratinib, CI = 0.29. (C) The cell cycle assay was performed in HR⁺/HER2-low breast cancer cells administered the triple combination of 125 nM neratinib, CDK4/6 inhibitor (100 nM, palbociclib), and endocrine therapy (100 nM, ET, fulvestrant). For HER2-low cells’ G1 phase: DMSO vs. ET + CDK4/6i, P = 0.024; DMSO vs. ET + CDK4/6i + neratinib, P = 0.0001. (D) Cyclin D1, CDK4, HER2, and EGFR protein expression levels in HR⁺/HER2-0 and HR⁺/HER2-low breast cancer cells, measured by western blot. *P < 0.05; **P < 0.01; ***P < 0.001; n = 3. ET: endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner Neratinib.