Unveiling transcriptional mechanisms of B7-H3 in breast cancer stem cells through proteomic approaches.

B7-H3, an immune checkpoint molecule, is prominently overexpressed in various solid tumors, correlating with poor clinical outcomes. Despite its critical role in promoting tumorigenesis, metastasis, and immune evasion, the regulatory mechanisms governing B7-H3 expression, particularly in cancer stem cells (CSCs), remain elusive. In this comprehensive study, we focused on breast CSCs to uncover the transcriptional regulators driving B7-H3 overexpression. Utilizing DNA affinity purification-mass spectrometry (DAP-MS) to analyze B7-H3 promoter regions, we identified a novel set of transcription factors, including DDB1, XRCC5, PARP1, RPA1, and RPA3, as key modulators of B7-H3 expression. Functional assays revealed that targeting DDB1 with nitazoxanide significantly downregulated B7-H3 expression, subsequently impairing tumor sphere formation and cell migration in breast CSCs. These findings not only elucidate the complex transcriptional network controlling B7-H3 expression but also open new avenues for developing targeted immunotherapies aimed at disrupting CSC-driven cancer progression.