An anoikis-related gene signature predicts prognosis and immunotherapy response, and identifies CCAR2 as a therapeutic target in triple-negative breast cancer

Background Triple-negative breast cancer (TNBC) has poor prognosis, largely due to its high rates of metastasis and recurrence. Anoikis plays a pivotal role in tumor metastasis; however, its role in TNBC remains elusive. Methods Cox regression analysis was performed on anoikis-related genes (ARGs) to develop a prognostic signature. A clinical nomogram was developed based on the prognostic signatures. Associations of the signature with the immunogenomic landscape and response to targeted therapy and immunotherapy were assessed. Key prognostic genes were identified using random survival forest analysis. To evaluate Cell cycle and apoptosis regulator 2 (CCAR2) expression and its independent prognostic role, we performed RT-qPCR, immunohistochemistry, Kaplan-Meier analysis, and multivariate Cox regression analysis. In vitro functional experiments were conducted to assess the effect of CCAR2 on anoikis, migration, invasion, and stemness. Bioinformatics analyses were utilized to evaluate the association between CCAR2 expression and the immunological landscape and immunotherapy response. Results A six-gene prognostic signature based on ARGs was constructed, stratifying patients into high- and low-risk groups with a significant difference in overall survival. The nomogram achieved good calibration and strong discriminatory accuracy. Additionally, the signature showed significant correlations with the immunogenomic landscape and predicted the response to both targeted therapy and immunotherapy. Random survival forest analysis identified CCAR2 as a key gene. In TNBC, upregulation of CCAR2 independently predicted a higher histological grade, metastasis, and poor overall survival. Its knockdown inhibits cell migration and invasion, suppresses stemness, and induces anoikis. Furthermore, CCAR2 is associated with an immunosuppressive tumor microenvironment in basal-like TNBC, fostering an immune-evasive phenotype that correlates with a poorer predicted response to immunotherapy. Conclusion We constructed an ARG-based prognostic signature for TNBC and identified CCAR2 as a key metastatic driver that confers anoikis resistance, immune evasion, and stemness. These findings may advance the prognostic stratification of TNBC and could highlight CCAR2 as a potential therapeutic target for TNBC.