Introduction: HER2+ (ErbB2+) breast cancer (BC) patients demonstrate a high incidence (30%) of brain metastases (BM). Regardless of the effectiveness of ErbB2 targeting therapies in the therapy of primary HER2+ BC, BM remains a fatal complication. Restricted drug permeability across the blood-brain barrier and specific tumor- and/or brain tumor microenvironment (TME)-derived factors determine the low effectiveness of ErbB targeted drugs in the brain. Neuregulin-1 (NRG-1) is a member of the EGF family which is commonly expressed by cells in the brain TME. NRG1 can bind to ErbB3 and/or ErbB4 receptors and potentially promote activation of alternative signaling pathways under ErbB2 inhibition.

Results: The newly established patient-derived HER2+ BC model (BCBM94) that metastasizes to the brain in mice forms well-circumscribed proliferative tumors that are vascularized and surrounded by activated astrocytes. BCBM94 cells are sensitive to the pro-apoptotic actions of the reversible EGFR/ErbB2 small molecule tyrosine kinase inhibitor Lapatinib. NRG1 mitigated cytotoxicity induced by Lapatinib, as shown by higher viability in WST-1 assays and the preserved ability for long-term cell propagation in clonogenicity assays. NRG-1 blocked Lapatinib-driven PARP cleavage and activation of the pro-apoptotic caspases-3/7 and caspase-9. NRG-1 also prevented Lapatinib-induced mitochondrial damage. rhNRG1 rescued phosphorylation of kinase-impaired ErbB3 under combined Lapatinib/rhNRG1 treatment suggesting the involvement of ErbB3 in maintaining the viability of BCBM94 under Lapatinib. The essential role of ErbB3 in the apoptosis inhibiting action of rhNRG1 was confirmed by siRNA-mediated silencing (KD) of the receptor. Upon ErbB3 knockdown, rhNRG1 was no longer able to attenuate the Lapatinib-mediated apoptosis in BCBM94 and this coincided with the mitigated rescue of Survivin and XIAP expression and BAD phosphorylation in BCBM94. These rhNRG1-mediated anti-apoptotic actions were also prevented with exposure to the multi-targeted PI3K-Akt and mTORC1/C2 inhibitor (PI-103) confirming the role of the ErbB3-Akt-mTOR signaling axis in the cell viability rescue.

Conclusion: The findings identify BCBM94 as a valuable brain metastasis cell model to study resistance mechanisms under ErbB2 inhibition and demonstrate an important role of NRG1 as a powerful brain TME-derived anti-apoptotic factor that can facilitate resistance of BC brain metastases to ErbB2 targeting therapies.