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

## TL;DR

This study explores a new treatment for HER2-positive breast cancer using a triple-targeting regimen and immunotherapy to improve survival and overcome resistance.

## Contribution

The study introduces a triple-targeting regimen combined with immunotherapy to address resistance in HER2-positive breast cancer.

## Key findings

- RANK+ HER2+ patients on triple-targeting therapy had significantly better disease-free survival.
- Combining immunotherapy with triple-targeting reduced cell viability in HER2+ breast cancer cells.
- Triple-targeting therapy reduced tumor volume and weight in HER2+ xenograft models.

## Abstract

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.