Abstract
Purpose: This study aims to explore the causal association between members of the cathepsin family and breast cancer subtypes, with a focus on their expression profiles, prognostic significance, and potential molecular mechanisms in HER2-positive and HER2-negative breast cancer. Additionally, the study includes preliminary in vitro cell experiments to validate the functional relevance of key genes. Methods: This study employs the two-sample Mendelian randomization (MR) method to analyze the causal relationship between 10 members of the cathepsin family (CTSB, CTSE, CTSF, CTSG, CTSH, CTSL1, CTSL2, CTSO, CTSS, CTSZ) and breast cancer risk. Sensitivity analyses, including MR-Egger, Cochran's Q test, and MR-PRESSO, are used to validate the robustness of the results. Additionally, the study integrates differential gene expression analysis, prognostic impact analysis, single-cell analysis, protein-protein interaction (PPI) network construction, and targeted cell experiments (qRT-PCR for gene expression, CTSO overexpression model construction, CCK-8 proliferation assay, and wound healing migration assay) to systematically investigate the role of cathepsins in breast cancer. Results: MR analysis identified significant associations between CTSE and CTSO expression with breast cancer risk. Elevated CTSE expression was positively associated with both HER2-positive (OR = 1.108, P = 0.022) and HER2-negative (OR = 1.099, P = 0.009) breast cancer risk. In contrast, CTSO expression was inversely associated with HER2-negative breast cancer risk (OR = 0.913, P = 0.037). Differential expression analysis confirmed that CTSE was overexpressed in HER2-positive tumors, while CTSO was underexpressed across breast cancer subtypes. Prognostic analysis showed that high CTSE expression was linked to a favorable prognosis in HER2-positive breast cancer patients treated with chemotherapy, whereas high CTSO expression correlated with improved prognosis in HER2-negative patients. Single-cell analysis revealed that CTSO was highly expressed in immune cells and fibroblasts, while CTSE was mainly localized to cancerous epithelial cells. PPI and functional enrichment analyses suggested that CTSE is involved in lysosomal and protein digestion pathways, whereas CTSO is implicated in immune regulation and antigen processing. Cellular experiments revealed that qRT-PCR did not detect quantifiable levels of CTSE in breast cancer cell lines, while CTSO exhibited low expression across all breast cancer cell lines. After overexpressing CTSO in MDA-MB-231 cells, CCK-8 assays showed a significant reduction in cell proliferation, and wound healing assays demonstrated a marked inhibition of cell migration. Conclusion: This study provides multi-layered evidence for the association between members of the cathepsin family (particularly CTSE and CTSO) and breast cancer subtypes through MR analysis, bioinformatics validation, and cellular experiments. The findings highlight the potential of these markers as subtype-specific biomarkers for precision diagnosis, treatment, and prognosis evaluation in breast cancer. These results deepen our understanding of the biological mechanisms underlying breast cancer and offer both experimental and theoretical support for the development of novel therapeutic strategies targeting the cathepsin family.