Investigating the immunological function of alpha-2-glycoprotein 1, zinc-binding in regulating tumor response in the breast cancer microenvironment

Background: The immunosuppressive function of androgen receptor (AR) in breast cancer is known; however, the mechanism remains unclear. Elucidating the immunoregulatory mechanisms in the breast cancer microenvironment will help develop novel treatment strategies. Here, we focused on AR-dependent secreted proteins encoded by the AZGP1 gene in breast cancer: alpha-2-glycoprotein 1, zinc-binding (ZAG), which is structurally similar to HLA class I and is implicated in immune regulation. Methods: We performed a gene set enrichment analysis (GSEA) to screen the biological processes associated with AZGP1 expression using a gene expression profile dataset of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Subsequently, we analyzed the correlation between AZGP1 expression and the immune cell composition in breast cancer tissues estimated with the CIBERSORTx using METABRIC and another dataset of The Sweden Cancerome Analysis Network- Breast. In our previous study of 45 breast cancer tissues, we evaluated the infiltration of 11 types of immune cells using flow cytometry (FCM). ZAG expression was further evaluated by immunohistochemistry, and the relationship between ZAG and the immune cells was analyzed. The action of ZAG in M1/M2 polarization models constructed using primary culture of human peripheral blood mononuclear cells (PBMC)-derived macrophage (Mφ) based on M1/M2 differentiation markers (Cluster of Differentiation (CD) 86, CD80/CD163, MRC1) and HLA class I/II expression evaluated by FCM (n=15 each). Results: GSEA demonstrated that AZGP1 expression was negatively correlated with multiple gene sets representing immunological processes. AZGP1 expression was inversely correlated with Mφ M1 infiltration estimated by CIBERSORTx. ZAG expression was associated with decreased infiltration of monocytes/macrophages, non-classical monocytes, and myeloid-derived suppressor cells in breast cancer tissues assessed by FCM. In in vitro analyses, ZAG decreased the expression of CD80, CD163, MRC1, and HLA classes I and II in the M1 polarization model and the expression of CD163 and MRC1 in the M2 polarization model. Conclusion: ZAG is a novel immunoregulatory factor affecting Mφ phenotype and possibly function in breast cancer tissues. Our findings lay the foundation for future studies to elucidate the immunological role of ZAG in breast cancer.