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Risk stratification and multi-omics reveal RAD51 inhibition as a radiosensitizing strategy in triple-negative breast
Ran An1,2,3,4, Jianming Li1,2,3,4, Tianhui Liu1,2,3,4
1Department of Breast Imaging, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China.
Background:
Triple-negative breast cancer (TNBC) is a highly aggressive subtype characterized by significant inter- and intra-tumoral heterogeneity. Its varied response to radiotherapy limits clinical outcomes, yet the underlying molecular determinants remain poorly understood.
Methods:
By integrating single-cell and bulk transcriptomic data, we profiled cell-type-specific radiosensitivity and defined distinct molecular subtypes via consensus clustering. A random survival forest (RSF) prognostic model was then constructed using 34 survival-associated genes and interpreted via SHAP analysis, and its performance was validated in multiple independent external cohorts. RAD51 was prioritized via Dependency Map (DepMap) analysis, and its functional role was validated in vitro and in vivo, supporting its role as a biological dependency in radiotherapy response. Moreover, tumor immune remodeling was further evaluated by immunofluorescence and protein analysis.
Results:
Single-cell analysis revealed that fibroblasts, macrophages, and proliferative cells harbored elevated radioresistance signatures compared to lymphoid lineages. Bulk profiling identified two distinct subtypes: Cluster A (immunosuppressive, radioresistant, poor prognosis) and Cluster B (immune-active, favorable survival). The RSF model robustly stratified patient risk, with high-risk tumors tightly linked to an immune-desert microenvironment. DepMap analysis nominated RAD51 as a core biological dependency. Importantly, RAD51 knockdown sensitized TNBC cells to irradiation by exacerbating DNA damage, triggering G2/M phase arrest, and driving apoptosis. Consistently, in vivo RAD51 silencing synergized with radiotherapy to significantly suppress tumor growth. Notably, this combination reshaped the immunosuppressive TME, driving robust CD8+ T cell infiltration, M1 macrophage repolarization, and adaptive PD-L1 upregulation.
Conclusion:
This integrative multi-omics study delineates the cellular and molecular landscape of TNBC radioresistance. The RSF model provides a robust tool for risk stratification, while identifying RAD51 as a potential therapeutic target to restore radiosensitivity and reverse immunosuppression, particularly in high-risk, immune-cold tumors.
Insights
Triple-negative breast cancer (TNBC) shows varied radiotherapy response due to heterogeneity. Targeting RAD51 may restore radiosensitivity and reverse immunosuppression in aggressive tumors.
Area of Science:
- Oncology
- Genomics
- Immunology
Background:
- Triple-negative breast cancer (TNBC) is aggressive and heterogeneous.
- Radiotherapy response in TNBC is variable and poorly understood.
- Molecular determinants of TNBC radiosensitivity require elucidation.
Purpose of the Study:
- To profile cell-type-specific radiosensitivity in TNBC.
- To define molecular subtypes and develop a prognostic model for TNBC.
- To identify therapeutic targets for improving radiotherapy response in TNBC.
Main Methods:
- Integrated single-cell and bulk transcriptomic analysis.
- Consensus clustering for molecular subtyping.
- Random survival forest (RSF) model construction and validation.
- Dependency Map (DepMap) analysis for target identification.
- In vitro and in vivo functional validation of RAD51.
- Immunofluorescence and protein analysis for immune microenvironment assessment.
Main Results:
- Fibroblasts, macrophages, and proliferative cells exhibit radioresistance.
- Two TNBC subtypes identified: Cluster A (immunosuppressive, radioresistant) and Cluster B (immune-active, favorable survival).
- RSF model accurately stratified patient risk, correlating high-risk with immune-desert tumors.
- RAD51 identified as a key dependency; its knockdown sensitized TNBC to irradiation.
- RAD51 inhibition combined with radiotherapy suppressed tumor growth and remodeled the tumor microenvironment (TME).
- Combination therapy enhanced CD8+ T cell infiltration, M1 macrophage repolarization, and PD-L1 expression.
Conclusions:
- Integrative multi-omics study reveals TNBC radioresistance mechanisms.
- RSF model offers robust risk stratification for TNBC patients.
- RAD51 is a promising therapeutic target to enhance radiosensitivity and overcome immunosuppression in high-risk TNBC.
