The Role of HER2 and RANK in Breast Cancer and New Therapeutic Approaches With Denosumab, Anti-HER2 Antibodies and Immunotherapy.

Despite advances in targeted therapies, resistance to anti-human epidermal growth factor receptor 2 (HER2) treatments remains a significant challenge in breast cancer (BC) management. This study aimed to evaluate the effectiveness of a triple-targeting regimen-Denosumab (D), Pertuzumab (P) and Trastuzumab (T)-for HER2-positive (HER2+) BC, and to assess the added value of immune checkpoint inhibitors (ICIs). Immunohistochemical analysis of 120 paraffin-embedded BC samples revealed that HER2+ tumours exhibited significantly higher receptor activator of nuclear factor (NF)-κB (RANK) expression compared to HER2-negative (HER2-) tumours. Notably, RANK-positive (RANK+)/HER2+ patients who received triple-targeting therapy experienced a statistically significant improvement in disease-free survival (DFS), while RANK-negative (RANK-)/HER2+ patients did not derive similar benefit. In BT-474 HER2+ xenograft mouse models, the combination of D+P+T significantly reduced tumour volume and weight. Additional analyses showed elevated signal transducer and activator of transcription 3 (STAT3) expression in HER2- tissues and higher mechanistic target of rapamycin (mTOR) expression in HER2- compared to HER2+ samples. Importantly, three-dimensional (3D) cell culture experiments demonstrated that adding ICIs (Nivolumab (N) and Ipilimumab (I)) to the triple-targeting regimen further reduced cell viability in HER2+ BC cells. These results underscore the pivotal role of the RANK-receptor activator of NF-κB ligand (RANKL) axis in tumour growth and immune regulation, supporting the use of triple-targeting therapy and suggesting enhanced benefits with the inclusion of ICIs to potentially overcome therapeutic resistance in HER2+ BC.