Neratinib enhances the efficacy of CDK4/6 inhibitor plus endocrine therapy in HR+/HER2-low breast cancer cell line ZR-75-1 via hsa-miR-23a-5p

Abstract

HR⁺/HER2-low breast cancer is a significant subgroup of conventional HR⁺/HER2-negative breast cancer, and combination of CDK4/6 inhibitor and endocrine therapy is the standard first-line and second-line treatments for advanced HR⁺/HER2-low breast cancer. Nevertheless, it remains uncertain whether HER2 signaling affects the effectiveness of CDK4/6 inhibitor administered in combination with endocrine therapy for HR⁺/HER2-low breast cancer and suitable intervention measures. This study revealed poor efficacy for CDK4/6 inhibitor combined with endocrine therapy for HR⁺/HER2-low breast cancer in vitro and in vivo models. Secondly, suppression of HER2 gene expression in HR⁺/HER2-low breast cancer cells resulted in significantly improved efficacy for CDK4/6 inhibitor combined with endocrine therapy. Furthermore, the anti-HER inhibitor neratinib was administered to enhance the effectiveness of CDK4/6 inhibitor combined with endocrine therapy in HR⁺/HER2-low breast cancer by inhibiting the HER2 pathway and lowering HER2 mRNA expression. Strikingly, neratinib reversed the efficacy of CDK4/6 inhibitor and endocrine therapy by reducing HER2 mRNA stability in HR⁺/HER2-low breast cancer through the interaction of HER2 3'-UTR region with hsa-miR-23a-5p. Even after reducing neratinib dosage to the standard 1/2 dose (20 mg/kg), it remained highly effective and well-tolerated. This study provides a viable and well-tolerated triple combination therapy for clinical HR⁺/HER2-low breast cancer.

Keywords: CDK4/6 inhibitor; Endocrine therapy; HER2 mRNA stability; HR+/HER2-low; Hsa-miR-23a-5p; Neratinib.

Fig. 1

Characterization of ZR-75-1 as an HR⁺/HER2-low breast cancer cell line. (A,B) Heat maps of gene expression in PAM50 cells of different breast cancer subtypes and heat maps of ERBB2 expression in HR⁺/HER2- breast cancer cells. (C) HER2 expression in HR⁺/HER2-0 and HR⁺/HER2-low breast cancer subtypes by cellular immunofluorescence assay. (D) HER2 levels in HR⁺/HER2-0 and HR⁺/HER2-low breast cancer subtypes measured with ImageJ, P = 0.029. (E) HER2 expression in HR⁺/HER2-0 and HR⁺/HER2-low breast cancer subtypes by fluorescence quantitative PCR assay, P = 0.015, *P < 0.05; n = 3.

Fig. 2

Efficacy of CDK4/6 inhibitor combined with endocrine therapy in the ZR-75-1 cell line in vitro and in vivo. (A,B) CCK-8 and colony formation assays were performed to detect the effects of various concentrations of CDK4/6 inhibitor (palbociclib) combined with endocrine therapy (100 nM, fulvestrant) on the proliferation of HR⁺/HER2-low breast cancer cells. The effect was not as significant as in HR⁺/HER2-0 breast cancer. For 200 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.003. For 400 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.0001. For 800 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.0001. For 100 nM CDK4/6i + 100 nM ET: HER2-low vs. HER2-0, P = 0.0001. (C–E) CDK4/6 inhibitor (palbociclib, 150 mg/kg, i.g.) combined with endocrine therapy (fulvestrant, 100 mg/kg, i.m.) inhibited tumor growth in HR⁺/HER2-low breast cancer xenografted mice, but the effect was not as significant as in the HR⁺/HER2-0 subtype. For HER2-low tumor volume on day 21: normal saline vs. ET + CDK4/6i, P = 0.001. For HER2-0 tumor volume on day 21: normal saline vs. ET + CDK4/6i, P = 0.0001. For tumor inhibition rate on day 21: HER2-low vs. HER2-0, P = 0.0001. (F) ER, PR, and HER2 expression levels in tumor tissues of HR⁺/HER2-0 and HR⁺/HER2-low breast cancer xenografted mice, measured by immunohistochemistry, the arrow points to the positive area and scale bar = 100 μm. **P < 0.01; ***P < 0.001; n = 6. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, ER: estrogen receptor, PR progesterone receptor.

Fig. 3

Role of HER2 in the poor efficacy of CDK4/6 inhibitor combined with endocrine therapy in HR⁺/HER2-low breast cancer. (A,B) Stable knockdown of HER2 in human HR⁺/HER2-low breast cancer cells by lentiviral shRNAs (shHER2 #1 and shHER2 #2). Knockdown effects were verified by western blot and quantitative PCR. shNC vs. shHER2 #1, P = 0.0009; shNC vs. shHER2 #2, P = 0.0019. (C,D) Knockdown of HER2 enhanced the effect of CDK4/6 inhibitor (250 nM, palbociclib) combined with endocrine therapy (100 nM, fulvestrant) in inhibiting HR⁺/HER2-low breast cancer cell proliferation as examined by the CCK-8 and colony formation experiments assays. For 100 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.004. For 200 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.045. For 400 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.019. For 800 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.001. For 1600 nM CDK4/6i + 100 nM ET: shNC vs. shHER2, P = 0.016. (E–H) In cell cycle assay, western blot, and apoptosis assay, knockdown of HER2 promoted G1 phase arrest and reduced Cyclin D1, CDK4 and EGFR expression. For HER2-low cells G1 phase: shNC vs. shHER2, P = 0.001. **P < 0.01; ***P < 0.001; n = 3. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor.

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.

Fig. 5

Neratinib enhances the efficacy of CDK4/6 inhibitor combined with endocrine therapy in HR⁺/HER2-low breast cancer in vivo. (A) Tumor photographs in animals treated with normal saline (control group), CDK4/6 inhibitor (palbociclib, 150 mg/kg, i.g.) combined with endocrine therapy (fulvestrant, 100 mg/kg, i.m.), and triple therapy consisting of standard dose of neratinib group (40 mg/kg, i.g.), CDK4/6 inhibitor, and endocrine therapy. (B) Weight changes in each group of HR⁺/HER2-low tumor-bearing mice. For HER2-low weight on day 14: normal saline vs. ET + CDK4/6i, P = 0.217; normal saline vs. standard dose ET + CDK4/6i + Ner, P = 0.0052. (C) Tumor growth curves and (D) tumor inhibition rates of HR⁺/HER2-low tumor-bearing mice in various groups. For HER2-low tumor volume on day 14: normal saline vs. ET + CDK4/6i, P = 0.009; normal saline vs. standard dose ET + CDK4/6i + Ner, P = 0.0003. For HER2-low tumor inhibition rate on day 14: normal saline vs. ET + CDK4/6i, P = 0.0055. (E) Tumor HER2 mRNA expression of in each group of HR⁺/HER2-low tumor-bearing mice. For HER2-low tumor HER2 mRNA expression on day 14: normal saline vs. ET + CDK4/6i, P = 0.381; normal saline vs. standard dose ET + CDK4/6i + Ner, P = 0.001. (F) HER2 expression in mouse HR⁺/HER2-low breast cancer tissue by IHC staining. (G) Cyclin D1, CDK4 and EGFR protein expression levels in HR⁺/HER2-low breast cancer, measured by western blot. *P < 0.05; **P < 0.01; ***P < 0.001; n = 5. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner neratinib.

Fig. 6

Neratinib enhances the efficacy of CDK4/6 inhibitor combined with endocrine therapy by reducing HER2 mRNA stability through the interaction of HER2’s 3’-UTR region with hsa-miR-23a-5p. (A,B) After knockdown of HER2, the effect of the triple combination of neratinib, CDK4/6 inhibitor, and endocrine therapy on HR⁺/HER2-low breast cancer cell proliferation was assessed by the CCK-8 and colony formation assays. (C,D) In cell cycle analysis and western blot, neratinib was shown to promote G1 phase arrest through the HER2 pathway, to downregulate Cyclin D1 and CDK4, and to improve the effect of CDK4/6 inhibitor combined with endocrine therapy in HR⁺/HER2-low breast cancer. (E) Effects of CDK4/6 inhibitor + endocrine therapy and triple combination treatment (neratinib, CDK4/6 inhibitor and endocrine therapy) on HER2 mRNA stability. MicroRNA sequencing (F) revealed the effect of neratinib on miRNA changes, and compared with CDK4/6 inhibitor + endocrine therapy group, the triple combination treatment group administered neratinib, CDK4/6 inhibitor, and endocrine therapy had significantly upregulated hsa-miR-23a-5p (G), which highly interacted with HER2’s 3’-UTR region (H), and significantly lowered HER2 mRNA stability (I) and downregulated HER2 and EGFR protein expression (J). *P < 0.05; **P < 0.01; ***P < 0.001; n = 3. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner neratinib.

Fig. 7

Optimization of neratinib dosing for enhanced CDK4/6 inhibitor combined with endocrine therapy efficacy. (A) Tumor photographs in animals treated with normal saline (control group), CDK4/6 inhibitor (palbociclib, 150 mg/kg, i.g.) + endocrine therapy (fulvestrant, 100 mg/kg, i.m.), medium-dose neratinib (20 mg/kg) alone, a triple therapy consisting of medium-dose neratinib group (20 mg/kg, i.g.), CDK4/6 inhibitor, and endocrine therapy, and a triple therapy consisting of low-dose neratinib (10 mg/kg, i.g.), CDK4/6 inhibitor, and endocrine therapy. (C) Weight changes in various groups of HR⁺/HER2-low tumor-bearing mice. (B) Tumor inhibition rates and (D) tumor growth curves of HR⁺/HER2-low tumor-bearing mice in various groups. For HER2-low tumor volume on day 21: normal saline vs. medium-dose ET + CDK4/6i + Ner, P = 0.0001; normal saline vs. low-dose ET + CDK4/6i + Ner, P = 0.0017; normal saline vs. ET + CDK4/6i, P = 0.003; ET + CDK4/6i vs. medium-dose ET + CDK4/6i + Ner, P = 0.0021. For HER2-low tumor inhibition rate on day 21: normal saline vs. medium-dose ET + CDK4/6i + Ner, P = 0.0217; normal saline vs. low-dose ET + CDK4/6i + Ner, P = 0.1271. ^**P < 0.01; ^***P < 0.001; n = 5. ET endocrine therapy, CDK4/6i CDK4/6 inhibitor, Ner neratinib.